An immunoglobulin G fraction from antiserum raised against cholesterol side-chain cleavage cytochrome P-450 (cytochrome P-450sec) purified from bovine adrenocortical mitochondria cross-reacted with human fetal adrenal cytochrome P-450sec in an Ouchterlony double diffusion system. This property of the immunoglobulin G fraction was used for the immunoisolation of cytochrome P-450sec of human fetal adrenal cells in culture. The molecular weight of the immunoisolate from [35S] methionine-labeled human fetal adrenal cells was similar to that of purified bovine adrenocortical cytochrome P-450sec (49,000). However the molecular weight of cytochrome P-450sec immuno-isolated from an in vitro translation system programmed by human fetal adrenal RNA was approximately 54,000, about 5,000 larger than that of the immunoisolate from radiolabeled cells. The rate of synthesis of cytochrome P-450sec by human fetal adrenal cells in culture, when incubated with ACTH (10(-6) M) for 36 h, was 2.5-fold greater than that by fetal adrenal cells incubated in the absence of ACTH. This result suggests that, as in the case of bovine adrenal cells, cytochrome P-450sec of human fetal adrenal tissue is synthesized as a larger molecular weight precursor which presumably undergoes processing to the mature form during translocation into the mitochondria. This synthesis is stimulated by ACTH, which is the putative trophic factor regulating human fetal adrenal steroidogenesis.