To examine whether mating can occur within as well as between clones of Trypanosoma brucei, we transformed three T. brucei subspecies stocks with heterologous genes conferring resistance to either hygromycin or Geneticin and carried out a series of inter- and intraclone matings in all possible double drug combinations. Double drug-resistant hybrids were recovered from three of the six out-crosses, but not from any of the three intraclone matings. However, further analysis of cloned progeny trypanosomes from one of the out-crosses using RFLP markers, molecular karyotyping and RAPD (random amplification of polymorphic DNA) produced unequivocal evidence that intra- as well as interclone mating had occurred. The progeny of interclone mating were double drug-resistant and heterozygous at 9 of 13 loci examined. In contrast, the progeny of intraclone mating had no demonstrable input of genetic material from the hygromycin-resistant parent and were similar to the Geneticin-resistant parent for most markers, except for five loci which were heterozygous in the Geneticin-resistant parent but homozygous in these clones (aldolase THT1 glucose transporter, procyclin, tubulin and cDNA 23). In addition, PFGE showed considerable karyotypic rearrangements in these clones and loss of genetic material was evident from RAPD and VSG (variant surface glycoprotein) gene fingerprint analysis. We conclude that intraclone mating can occur in trypanosomes, but only during out-crossing, suggesting that meiosis and/or fusion are triggered by a diffusible factor.