The frequency of hybrid formation between two Trypanosoma brucei clones during cyclical transmission through Glossina morsitans centralis was analyzed. In two independent experiments, teneral G. m. centralis were infected with an equal mixture of two T. brucei clones showing different homozygous isoenzyme patterns for isocitrate dehydrogenase (ICD; E.C.1.1.1.42) and alkaline phosphatase (AP; E.C. 3.1.3.1). Trypanosomes were cyclically transmitted to mice from 23 infective flies and the subsequent bloodstream-form populations were characterized by isoenzyme electrophoresis. Heterozygous patterns for ICD and AP indicated that hybrid formation occurred in at least 9 of the 23 vectors. There was further evidence that extrusion of hybrid parasites with saliva from a single fly was not necessarily continuous but could alter over time with the occurrence of either or both of the homozygous parental clones.