Kinetoplastids are unicellular eukaryotes that include important parasites of man, such as trypanosomes and leishmanias. The study of these organisms received a recent boost from the development of transient transformation allowing the short-term expression of genes reintroduced into parasites like Trypanosoma brucei, the causative agent of African trypanosomiasis. We have obtained long-term stable transformants of T. brucei that have acquired the ability to grow in medium containing the drug G418, following the targeted insertion of the bacterial gene for neomycin phosphotransferase (neo(r) gene) into the trypanosome tubulin cluster. Plasmids in which part of the T. brucei tubulin gene cluster has been replaced by the neo(r) gene were used. Targeting efficiency was higher with a linearized than with a circular construct, and with 5 kilobases of tubulin gene cluster than with 0.9 kilobases. With these neo(r) constructs homologous recombination seems to be the preferred route for insertion of exogenous DNA into the trypanosome genome, allowing gene targeting without counter-selection.