The present study further examined the role of arachidonic acid (AA) in the action of LHRH on ovarian steroidogenesis. Rat granulosa cells were incubated with LHRH and/or AA in the presence of FSH for 24 h. As expected, the presence of LHRH markedly attenuated the FSH-induced progesterone (P) production. AA by itself did not affect the magnitude of P production induced by FSH. However, when AA (10(-5) M) was added during the last 6 h to the cultures containing LHRH and FSH, AA partially reversed the inhibitory action of LHRH (by 42%). Likewise, addition of AA partially antagonized the inhibitory effect of 12-O-tetradecanoylphorbol-13-acetate (TPA; 10(-9) M) on FSH-induced P production (by 71%). Pretreatment of granulosa cells with TPA or LHRH without FSH for 18 h did not alter the stimulatory effect of AA on P formation during the subsequent 6-h culture period. In other experiments granulosa cells were incubated for 5 h in the absence of FSH. Treatment with AA, TPA, or LHRH alone caused significant increase in P production. Combined treatment with AA and LHRH or AA plus TPA showed additive effects on P formation. By contrast, AA failed to potentiate the effect of LHRH or TPA on 20 alpha-hydroxyprogesterone accumulation. Although AA by itself slightly stimulated 20 alpha-hydroxyprogesterone production, the magnitude was much less than that induced by TPA or LHRH. When 25-hydroxycholesterol was present in the incubation medium, P production was significantly increased. In the presence of 25-hydroxycholesterol, AA further increased P formation, but did not enhance the stimulatory actions of LHRH or TPA in this regard. Taken together, these results further support the hypothesis that AA (or its metabolites) play a stimulatory role in the direct action of LHRH on ovarian steroidogenesis.