Maturation of the precursor forms of bovine cholesterol side-chain cleavage cytochrome P-450 (P-450SCC) and 11 beta-hydroxylase cytochrome P-450 (P-450(11)beta) was investigated using mitochondria from bovine corpus luteum. The results show that both precursors, whose synthesis was directed by bovine adrenocortical RNA, can be imported and proteolytically processed to their corresponding mature forms by bovine corpus luteal mitochondria, even though P-450(11)beta is not expressed in this tissue. Furthermore, the efficiency of processing of pre-P-450(11)beta by corpus luteal mitochondria is similar to that of pre-P-450SCC, an endogenous enzyme of these mitochondria. However, the P-450(11)beta precursor is not processed by mitochondria from a nonsteroidogenic tissue (heart), a result observed previously for the P-450SCC precursor (M. F. Matocha and M. R. Waterman (1984) J. Biol. Chem. 259, 8672-8678). This discriminatory processing of pre-P-450(11)beta by heterologous mitochondria suggests that the precursor forms of P-450SCC and P-450(11)beta are processed via a common pathway in steroidogenic mitochondria and that this pathway is absent in nonsteroidogenic mitochondria.