Capturing and holding of white rhinoceros (Ceratotherium simum) for the reintroduction to new reserves or breeding in zoos often involves a risk of mortality. Non-invasive techniques to monitor the stress experienced by these animals may guide the selection of management techniques that reduce risks to animal well-being. The aim of the study was to evaluate the biological relevance of a developed technique to monitor stress hormone metabolites in faecal samples of wild-caught and captive-bred white rhinoceros. Faecal corticosterone concentrations were measured via radioimmunoassay (125I RIA), in seven white rhinoceros (3 males and 4 females), at three sites, before and after an adrenocorticotropic hormone (ACTH) challenge test and control saline injection. Administration of ACTH resulted in a significant increase in faecal corticosterone concentrations (up to 350% above pre-ACTH baseline) within 1-2 days of injection, returning to baseline 4 days post-injection. It was found that individual baseline corticosterone concentrations fluctuate naturally and vary between individual animals, suggesting that an adequate baseline period of faecal sampling is needed in order to accurately assess responses to ACTH stimulation. Furthermore, the technique proved sensitive enough to detect elevations in faecal corticosterone concentrations due to environmental stressors. Data of faecal corticoid concentrations were correlated with gastrointestinal transit (GIT) times before and after ACTH and saline treatment by using art glitter as a digestive marker. This showed that gut passage times correlated to the ACTH-induced time to peak. Overall the results confirm that measurements of faecal corticosterone metabolites with the validated 125I RIA is a useful diagnostic tool to monitor adrenocortical activity in white rhinoceros. This study can therefore provide a methodology for examining chronically heightened adrenal activity in these animals and consequently be used to inform management strategies that aim to improve the welfare of white rhinoceros in captivity.