The aim of this study was to standardize and validate an immunohistochemical test for the routine diagnosis of African horsesickness in horses. Hamblin developed the primary anti- African horsesickness virus serum that I used and the avidin-biotin complex detection system was employed. During the standardization process I demonstrate that lung, heart and spleen samples are the most reliable. I also show that it is not necessary to take multiple samples per organ, because the AHSV-positive signal is generally widespread throughout the lung and heart, in particular. In order to validate the technique, samples from 118 negative and 128 positive horse cases, including all nine known serotypes, were immunostained. All of the positive cases were confirmed by means of virus isolation. Negative horse samples were obtained from countries where African horsesickness does not occur. None of the negative cases stained positive and all the positive cases were correctly identified. Therefore, there was 100 % concordance between immunohisto chemistry (when applied to formalin-fixed, paraffin-embedded heart and/or lung and/or spleen tissues from positive horse cases that had been archived for less than 10 years) and virus isolation results. Heart and lung had consistently more positive signal than spleen. The Hamblin antiserum did not cross-react with closely-related orbiviruses (specifically equine encephalosis virus and bluetongue virus) in selected horse and sheep tissues, respectively. Characteristic positive staining was observed in lung, heart and spleen samples from two dogs that died of African horsesickness. Positive signal was not affected by long-term storage in formaldehyde (up to 365 days). Also, specific positive staining could be detected in heart and/or lung and/or spleen samples in more than 95 % of positive horses where tissue blocks had been stored for between 10 and 83 years. The principal target cells in the horse and dog cases were microvascular endothelial cells, intravascular monocyte-macrophages and, to a lesser extent, interstitial macrophages in lung, spleen and liver, in particular. Positive staining is intracytoplasmic with a bead/dot and/or granular character. Beads, dots or granules may occur singly or in clusters. Occasionally, linear deposits of positive signal delineate segments of capillary vessels. The veterinary pathologist must look for characteristic positive signal in target cells, because, occasionally, certain bacteria (Rhodococcus equi and Helicobacter sp.) cross-react with the Hamblin antiserum. Clearly, the test is highly sensitive, specific and robust, sufficiently so for the routine diagnosis of African horsesickness virus.