Human adenoviruses (HAdVs) are non-enveloped DNA viruses, currently comprising 52 serotypes which are divided into seven species, designated A to G. The HAdVs are associated with a number of diseases affecting respiratory, urinary, gastrointestinal tracts and the eye. Human AdVs have increasingly been recognized as important pathogens in immunocompromised individuals. Human AdVs are ubiquitous in the environment resulting in the possible contamination of treated and untreated drinking water supplies by human secretions and excretions. As AdVs do not have an envelope, they are extremely resistant to inactivation, allowing for prolonged survival in the environment. The presence of AdVs in water sources is considered important, as they are exceptionally resistant to selected water treatment processes. Precise typing of HAdVs is, therefore, essential for epidemiological surveillance and the understanding of infection chains. The aim of this study was to determine the prevalence and genetic heterogeneity of HAdVs circulating in communities in selected regions of Africa compared to the rest of the world. It is also important to determine the genetic relationship between HAdVs strains occurring in water sources and those detected in human clinical specimens, as this may give some indication as to whether or not water sources are a potential source of infection. As part of ongoing surveillance in southern Africa of treated and untreated water sources for enteric viruses, 765 water samples were tested using a nested polymerase chain reaction (nPCR) for HAdVs. Of these samples, 65 (8.6%) water samples were positive for HAdVs, and selected samples were characterised. In the untreated water, HAdV-F was the dominant species (65.6%) and HAdV-D was second-most common (21.9%) species identified. Species HAdV–B, -A and –C were identified amongst the rest of the strains. From treated water, HAdV-D and –F were identified in one isolate each. Analysis of diarrhoeal stool specimens for HAdVs identified HAdV-F as the predominant species, comprising 77.8% of the identified strains, with species HAdV-C and –A less common, identified in 11.1% specimens. In the respiratory specimens from the same region, HAdV-C was identified in 28.6% of the specimens. Comparative genetic analysis of HAdVs from water sources and clinical specimens showed genetic relatedness between the strains. Water may therefore play an important role as source of infection in the surrounding communities. In developing countries, diarrhoea is a major cause of morbidity and mortality and after rotaviruses HAdVs are considered to be the second-most important cause of viral infantile diarrhoea. Samples also were available from Kenya, where there are very little data on the prevalence and distribution of HAdV serotypes associated with diarrhoea in paediatric patients. From Kenya, 278 stool specimens were analysed, of which 104 (43 diarrhoea; 61 non-diarrhoea) were from an urban hospice for human immunodefiency virus (HIV)-seropositive children, 94 from selected urban clinics and 80 from the rural setting. From these, the detection of HAdVs in diarrhoeal and non-diarrhoeal stool specimens was 43.3% and 16.4%, respectively. In the urban hospice setting, 43.3% of the stool specimens from HIV-seropositive children tested positive for HAdV. The overall detection of HAdVs species and genotypes in the stool specimens showed HAdV-D to predominate, being detected in 36.1% of specimens with HAdV-C (29.5%), HAdV-F (16.4%), HAdV-B (13.1%), and HAdV-A (6.5%) present in lower numbers. This study provided valuable new data on the prevalence and distribution of HAdV genotypes in diarrhoeal stool specimens in Africa. In this study where nucleotide sequence comparison was used to determine the genetic relatedness of African HAdVs to those from the rest of the world, it was noted that in most cases the African strains differed from those from the rest of the world. The use of molecular techniques for the detection and characterisation of HAdVs, especially in Kenyan cohorts, was of importance, as it provided new baseline data for further burden of disease studies which are necessary for future prevention and treatment programmes.