Leishmania mexicana amazonensis amastigotes were induced to differentiate by incubation at 27 C. Morphological transformation was studied both in untreated cultures and in cultures where DNA synthesis, and consequently the final stage in the production of promastigotes, was inhibited by hydroxyurea. 3-Methoxybenzamide and other antagonists of ADP-ribosyltransferase (ADPRT) specifically inhibited differentiation at a very early stage in both experimental systems. Cell proliferation (in the absence of hydroxyurea) was not inhibited by ADPRT antagonists--indeed greater multiplication of undifferentiated parasites was observed in the presence of these compounds. This indicated that the parasites were being diverted from differentiation to proliferation. Preincubation of the amastigotes with the ADPRT antagonists was required to produce this effect, providing further evidence that ADP-ribosylation of proteins is required for the initiation of differentiation in Leishmania.