The effects of the polyunsaturated fatty acids (PUFAs)--eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), and arachidonic acid (AA)--on in vitro steroid production by full-grown prematurational ovarian follicles from goldfish and rainbow trout were investigated. EPA and DHA inhibited gonadotropin-stimulated testosterone production in a dose-related manner, but AA was inhibitory only at the highest dose tested (400 microM). AA alone stimulated testosterone production by increasing cAMP production, but the effects of other PUFAs alone were marginal. The inhibitory actions by PUFAs were not restricted to long-chain PUFAs, as linoleic and linolenic acids had similar actions in the goldfish. The inhibitory action of EPA on testosterone production was reversible upon removal of the PUFA from medium. Testosterone production stimulated by the addition of the cAMP analogues, dibutyryl cAMP, and 8-bromo cAMP, was attenuated by PUFAs, suggesting that they act at a site distal to cAMP formation. A post-cAMP site regulating cholesterol availability may be involved as testosterone production induced by addition of 25OH-cholesterol was not affected by the PUFAs in either fish species. Together, these findings underscore the importance of lipids in ovarian physiology and suggest that PUFAs may participate in the regulation of ovarian steroidogenesis in teleost fish.