Our previous investigations demonstrated that 5- and 15-lipoxygenase metabolites of arachidonic acid are synthesized in bovine adrenal fasciculata cells, although their exact role in the regulation of adrenal steroidogenesis is unknown. Thus we attempted to investigate their direct effects on cholesterol side-chain cleavage in bovine adrenal mitochondria. We also examined Ca2+ efflux in mitochondria, based on a reported correlation between pregnenolone formation and Ca2+ release in adrenal mitochondria. The present experiments showed that 5-HETE increased pregnenolone generation in the isolated intact mitochondria, but not in the inner mitochondrial membrane. Thus it is suggested that 5-HETE may activate cholesterol side-chain cleavage by inducing the translocation of cholesterol from the outer to the inner mitochondrial membrane. The present experiments also demonstrated that 5-HPETE, 5-HETE, 15-HPETE, and 15-HETE, but not leukotriene B4, activated cholesterol side-chain cleavage and Ca2+ efflux from mitochondria, suggesting that these substances may activate cholesterol side-chain cleavage by regulating Ca2+ movement in mitochondria. These effects were additively enhanced when mitochondria were stimulated simultaneously with these substances and GTP. Therefore, both GTP and lipoxygenase metabolites seem to play crucial roles in the regulation of pregnenolone generation. The direct effect of 5- and 15-lipoxygenase metabolites other than leukotriene B4 on the regulation of pregnenolone synthesis, which is known to be the rate-limiting step of steroidogenesis, were clearly observed in the present experiments.