The mitochondrial matrix enzyme ornithine transcarbamoylase (OTCase; ornithine carbamoyltransferase; carbamoylphosphate:L-ornithine carbamoyltransferase, EC 2.1.3.3) is encoded by a nuclear gene on the X chromosome, synthesized on cytoplasmic ribosomes, and translocated across both mitochondrial membranes. Using specific immunoprecipitation, we presented evidence previously that the primary in vitro translation product of OTCase in rat liver is a polypeptide about 4000 daltons larger than the "mature" OTCase augment subunit purified from homologous mitochondria. In this report we augment the immunological identification of this cell-free translation product (pOTCase) with structural information and show, by electrophoresis of proteolysis products, that pOTCase is structurally similar to mitochondrial OTCase. Moreover, we now demonstrate that, when pOTCase is incubated posttranslationally with isolated rat liver mitochondria, it is converted to the size of mature OTCase and is sequestered within the mitochondria in such a way that it becomes resistant to externally added proteases. Such posttranslational processing is catalyzed specifically by the mitochondrial fraction of rat liver cells and is dependent both on the duration of incubation with mitochondria and on the amount of mitochondrial protein added. We conclude that pOTCase is indeed the bona fide precursor of mitochondrial OTCase and that use of this simplified cell-free system will facilitate analysis of OTCase biogenesis at both the cellular and the molecular level.