An immunoperoxidase assay for the detection of African horse sickness virus (AHSV) in formalin-fixed tissues is a valuable tool in the study of the pathogenesis of the disease, as well as a useful addition to existing diagnostic tests when only preserved tissues are available. An assay that uses Hamblin antiserum in a basic avidin-biotin complex detection system was standardized and validated in accordance with the guidelines of the American Association of Veterinary Laboratory Diagnosticians Subcommittee on Standardization of Immunohistochemistry. Using 128 positive cases of African horse sickness confirmed by viral isolation and serotyping and 119 negative cases from countries where the disease has never occurred, diagnostic sensitivity and diagnostic specificity were 100% in the prime target tissues of heart and lung. There was no variation in the ability of the assay to detect all 9 serotypes of AHSV, and there was no cross-reactivity with other orbiviruses in formalin-fixed tissues. The only cross-reactivity observed was in the lungs of 2 negative cases infected with Rhodococcus equi. The assay gave good results on tissues that had been fixed in formalin for up to 365 days. Nonspecific staining was minimal provided that the standard procedures for processing and staining tissues were followed. Good immunohistochemical results were also obtained on samples fixed as long as 24 hr after death. The assay, therefore, provides a robust diagnostic tool for detection of AHSV in formalin-fixed tissues, provided the analysis is done by an experienced pathologist.