Two groups, comprising 4 donkeys and 4 mules (group 1) and 4 donkeys and 3 mules (group 2), were used to determine the duration of viraemia and to monitor the development of antibodies following inoculation with African horse sickness virus (AHSV). One group of animals was given a single dose of attenuated AHSV serotype 4 (AHSV 4) vaccine. The second group was inoculated with a virulent field strain of AHSV 4. Both groups were subsequently challenged with the virulent field strain of AHSV 4, 51 and 58 days, respectively, after their primary inoculation. Blood and serum samples, collected on alternate days after the primary inoculations and also after subsequent challenge, were assayed for virus and antibodies. Seven of the 8 AHSV vaccinated (group 1) and 7 of the 7 AHSV inoculated (group 2) animals showed humoral antibody responses after primary inoculation. Although no infectious virus could be isolated from either group for the duration of the study, reverse transcription-PCR data obtained for the second group did show the presence of AHSV viral RNA from as early as day 5 in mules and day 9 in donkeys after the primary inoculation. Viral RNA was detected consistently up to day 47 in some animals and intermittently thereafter. There was no evidence of a second viraemia in any of the animals after challenge. The detection of specific antibodies, against AHSV 4 NS3 protein, in all animals confirmed that both donkeys and mules were infected and that the virus had replicated.