In many previous characterization studies of Trypanozoon, isolates have been subpassaged numerous times in laboratory rodents until a quantity of trypanosomes sufficient for analysis has been obtained. In addition to the numerous biochemical effects of such a process on the parasite, it appears probable that adaptation to an unnatural host may also serve to filter out less virulent populations from mixed infections, leading to an underestimate of the true level of genetic diversity. By the early cloning of trypanosomes from susceptible captive flies infected from the primary isolate--the midgut of a wild tsetse--the present study provides evidence of the range of genetically different Trypanosoma brucei populations which may coexist within the midgut of individual tsetse flies in nature. The three primary isolates from tsetse yielded one, five and nine genetically distinct populations. Cloned populations were confirmed as T. brucei using the polymerase chain reaction, and were characterized by karyotype analysis and multilocus isoenzyme electrophoresis. These data allowed a limited assessment of the level of genetic variability in natural populations of T. brucei.