The question whether tsetse flies can be experimentally infected with more than one trypanosome species or strain by sequential feeding was investigated using DNA probe technology to identify directly the small numbers of trypanosomes in the fly gut. Bloodstream form trypanosomes of Trypanosoma congolense or T. brucei ssp. were used for initial infection, followed by sequential feeds using either T. congolense or T. brucei ssp. Midgut trypanosome populations were subsequently analysed by hybridising dot blots with species-specific DNA probes. Two different T. brucei stocks were also fed in succession and the midgut trypanosome populations analysed by molecular karyotype. Contrary to expectations from previous reports, it was comparatively easy to superinfect flies with a second trypanosome species or stock, although the presence of trypanosomes already in the gut did not aid establishment of those incoming. Thus, to develop a mixed infection, a prerequisite for trypanosome mating, flies do not necessarily have to pick up both parental trypanosomes on their first feed.