Maturation-inducing steroid in amago salmon (Oncorhynchus rhodurus) has been identified from media in which immature but fully grown folliculated oocytes of amago salmon had been incubated for 18-24 hr with chum salmon gonadotropin (SGA). The maturation-inducing (MI) activity of residues at various steps of purification was assessed by an in vitro germinal vesicle breakdown (GVBD) assay based on fully grown prophase-arrested folliculated oocytes of amago salmon. Ether extracts of the media from these incubates showed high MI activity. Yolk and oil droplets were removed from the ether extract by partition with equal volumes of 50% methanol and n-hexane. MI activity was found only in the 50% methanol phase. The 50% methanol phase was then fractionated (20 separate fractions) by reversed-phase high-performance liquid chromatography. MI activity was found only in fraction 10 which had a retention time coinciding exactly with 17 alpha,20 beta-dihydroxy-4-pregnen-3-one (17 alpha,20 beta-diOHprog). The purity and final characterization of the residues of fraction 10 were further confirmed by thin-layer chromatography and mass spectrometry with authentic 17 alpha,20 beta-diOHprog standard. The present study, together with our previous findings that in amago salmon 17 alpha,20 beta-diOHprog is the most potent steroid for the induction of oocyte maturation in vitro and is present at high concentrations in the plasma only around the time of oocyte maturation, indicates that 17 alpha,20 beta-diOHprog is the major naturally occurring maturation-inducing steroid in this species.