A mitochondrial extract from Leishmania tarentolae directs the incorporation of uridylate (U) residues within the pre-edited domain of synthetic cytochrome b (CYb) and NADH dehydrogenase subunit 7 mRNA. This has several characteristics of an in vitro RNA editing activity, but no direct evidence for involvement of guide RNAs was obtained. Inhibition by micrococcal nuclease suggests a requirement for some type of endogenous RNA. The limitation of internal U-incorporation to the pre-edited region in the CYb mRNA and the inhibition by deletion or substitution of both mRNA anchor sequences for CYb gRNA-I and -II could be consistent either with a gRNA-mediated process or a secondary structure-mediated process. A low level of incorporation of [alpha-32P]CTP occurs at the same sites as UTP. Internal U-incorporation activity is selectively inhibited by heterologous RNAs, suggesting an involvement of low affinity RNA-binding proteins which can be competed by the added RNA.