The requirement of protozoan parasites for pre-formed purines affords the opportunity for quantitation of nucleic acid synthesis from incorporation of radioactively labeled purines into DNA and RNA. We have developed rapid and simple assays to quantitate DNA and RNA synthesis in promastigotes of Leishmania major and L. donovani from the incorporation of [3H]hypoxanthine. DNA but not RNA synthesis in L. major or L. donovani promastigotes was inhibited by aphidicolin (50% inhibition by 0.2-0.3 microM) and by hydroxyurea (50% inhibition by 0.3-0.5 mM). The inhibition of DNA synthesis by aphidicolin or hydroxyurea was reversible when the inhibitor was removed 2, 4 or 24 h after its addition. Several well-characterized agents that inhibit DNA synthesis in mammalian cells, 1-beta-D-arabinofuranosylcytosine (araC), 9-beta-D-arabinofuranosyladenine (araA), phosphonoacetic acid, novobiocin and N2-(p-n-butylphenyl)guanine (BuPG), failed to inhibit DNA synthesis in promastigotes of L. major even when used at very high concentrations, demonstrating differences between DNA replication components of parasite and host.