The persistence of sleeping sickness (human African trypanosomiasis) in some areas of south-eastern Uganda has necessitated further investigations, focusing mainly on domestic animals as reservoirs of this disease in three agro-ecological zones. The inter-zone differences in the prevalences of trypanosome infection among cattle (P < 0.001) and pigs (P < 0.001) were significant. Overall, 5.0% of the cattle, 13.9% of the pigs and 0.4% of the small ruminants investigated were found to be infected with parasites of the Trypanosoma brucei subgroup. The results of blood incubation infectivity tests (BIIT) indicated that all of the T. brucei-subgroup isolates from cattle in Kamuli district (zone I) were human-serum-sensitive. Of the zone-I pigs found infected, however, almost all (82.5%) were considered to be infected with T. brucei and many (30.2%) carried human-serum-resistant T. brucei. Pig-tsetse-human appears to be a major transmission cycle in zone I. In Mukono district (zone II), 10.5% and 26.1% of the T. brucei isolates from cattle and pigs, respectively, were human-serum-resistant, indicating that cattle-tsetse-human and pig-tsetse-human are major transmission cycles in zone II. In Tororo district (zone III), 47.3% of the T. brucei isolates from cattle were human-serum-resistant but there were no T. brucei isolates from pigs, indicating that cattle-tsetse-human is the major transmission cycle. Interestingly, as the only T. brucei isolate from sheep in zone III was human-serum-resistant, there may also be a sheep-tsetse-human cycle. In south-eastern Uganda, control efforts must be designed to eliminate the parasites not only from cattle but also from pigs and small ruminants.