The parasitic protozoan Leishmania mexicana differentiates from a non-motile intracellular amastigote in the mammalian macrophage phagolysosome into a motile, extracellular promastigote in the insect vector gut. This developmental program has been accomplished in vitro, thus providing a useful model for studying changes in the cytoskeleton during cell differentiation. The role of microtubules in leishmania differentiation was demonstrated by using the dinitroaniline herbicide oryzalin, which inhibited both leishmania proliferation and differentiation; 25 microM oryzalin reduced promastigote division by over 95%. Interestingly, at a sublethal dose (5 microM), promastigotes became round and multiflagellated but remained motile. At 50 microM oryzalin, the number of intracellular amastigotes decreased by 50%. However, leishmania differentiation seemed to be the most drug-sensitive stage: there was a 60% reduction in amastigote-to-promastigote differentiation at 0.5 microM oryzalin. The specific action of oryzalin on leishmania microtubules was verified by its inhibition of in vitro polymerization of leishmania microtubules, but not control mammalian microtubules (from rat brain). These findings indicate that microtubules play a major role in leishmania proliferation, maintenance of cell shape, and cytodifferentiation.