Genetic exchange in Trypanosoma brucei ssp. can occur when 2 different strains are cotransmitted through the tsetse fly vector. We have introduced heterologous genes for drug resistance (neo or hph) into parental trypanosome lines by electroporation. Drug resistant lines were then crossed in vivo in the fly or in vitro. Hybrids were subsequently selected by double drug resistance. Analysis of trypanosomes from both fly midguts and salivary glands showed the latter to be the probable site of genetic exchange. This is one of the first applications of reverse genetics to a longstanding problem in parasite biology.