Abstract Azaserine decreased the incorporation of 14C-labeled adenine, guanine, hypoxanthine, uracil, and orotic acid into the nucleic acid fraction and increased the incorporation of these compounds into the acid-soluble fraction of Trypanosoma equiperdum incubated in vitro. The amino acids l-glutamine, l-tryptophan, l-leucine, and l-tyrosine partially prevented the inhibition produced by azaserine on the incorporation of ade- nine- 14C into the nucleic acid fraction. Azaserine did not interfere with the incorporation of adenine- 14C into the acid-soluble adenine- and guanine-containing compounds but did inhibit the incorporation of uracil- 14C into acid-soluble cytosine-containing nucleotides and the UMP and CMP components of RNA. These results suggest that in T. equiperdum azaserine inhibits the conversion of UMP to CMP, and as a result of this inhibition there is a secondary inhibition of the biosynthesis of RNA. The extremely limited ability of T. equiperdum to anabolize tritiated cytosine or cytidine suggests that this protozoan species depends primarily upon the de novo pathway for the biosynthesis of CMP and indicates a possible site for exploitation with the appropriate antimetabolites.