We describe the isolation of a steroidogenesis-inducing protein (SIP) from human ovarian follicular fluid and its effects on in vitro steroid synthesis of testicular, ovarian, and adrenal cells. After heating at 60 C, precipitation with 80% ammonium sulfate and dialysis, the human ovarian follicular fluid proteins were fractionated by gel chromatography on Sephacryl S-200. The bioactivity was eluted in the mol wt region between 40 and 60 K. SIP was further purified by affinity chromatography on blue Sepharose (CL-6B), preparative isoelectrofocusing on sucrose density gradients, anion exchange chromatography on Mono Q by using fast protein liquid chromatography system and gel chromatography on Superose 12. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of purified SIP under both reducing and nonreducing conditions revealed a single band with an approximate mol wt of 60 K. SIP exhibited a pI value of 4.8, was heat sensitive, and lost activity upon lyophilization. SIP copurified with albumin in various isolation procedures and was distinct from human serum albumin but may represent a modified form of human albumin. SIP stimulated testosterone production by testicular pieces, interstitial cells, or purified Leydig cells from rats under different in vitro conditions. The SIP also stimulated basal and human CG stimulated in vitro progesterone production of human ovarian granulosa-lutein cells and corticosterone production of rat adrenal cells. The effects of SIP on testicular, ovarian, and adrenal cells were evident in the presence of maximal concentrations of tropic hormones. The steroid-free spent media from human granulosa-lutein cell cultures also stimulated testosterone production by rat interstitial cells, suggesting that granulosa cells may be the cellular source of SIP. In conclusion, human ovarian cells secrete a hitherto unknown albumin-like protein which enhances both basal and tropic hormone stimulated steroidogenesis in gonadal and adrenal cells. This protein may play a significant role in ovarian function by modifying steroidogenesis in the preovulatory follicle.