A severe influenza outbreak occurred in horses in South Africa in 1986. The causative agent was identified as an influenza virus [A/Equine/Johannesburg/86 (H3N8)]. Antigenic analyses of the hemagglutinin (HA) with ferret antisera and monoclonal antibodies showed that the Eq/Johannesburg/86 virus is similar to recent equine H3 viruses. The nucleotide sequence analysis on the HA genes of Eq/Johannesburg/86 and other equine H3 influenza viruses, together with the epidemiological data, clearly demonstrated that the Eq/Johannesburg/86 virus was derived from a virus that had been circulating in horses in the United States in 1986-87. The epidemiological information suggests that the unusually severe influenza outbreak in South Africa may be due to the lack of immunity to these viruses in the horse population.