Accumulation of cyclic GMP in estradiol-treated immature guinea pig myometrium was enhanced by carbachol, ionophore A 23186, unsaturated fatty acids and their hydroperoxides. Cyclic AMP content was elevated only by arachiodonic acid, A 23187 and PGI 2. Eicosatetraynoic acid (TYA), but not indomethacin prevented all cyclic GMP responses. The effects of A 23187 and arachidonate on cyclic AMP were accompanied by a parallel increase (2–3 fold) on the generation of PGI 2 by the myometrium. Both events were similarly reduced by indomethacin, TYA, 15-hydroperoxyarachidonic acid and tranylcypromine, suggesting that PGI 2 was involved. Omission of Ca 2+ or addition of mepacrine of p-bromophenacylbromide abolished the stimulatory effects of A 23187 and carbachol on cyclic GMP as well as the A 23187-induced elevations in both PGI 2 and cyclic AMP generation. Thus, with both exogenous arachidonate as well as with endogenous fatty acid, released through an apparent phospholipase A 2-induced activation process, the lipoxygenase pathway was associated with an activation of the cyclic GMP system and the cyclooxygenase pathway, via PGI 2 generation, with an activation of the cyclic AMP system. Carbachol failed to alter both cyclic AMP content and the release of PGI 2 suggesting a cholinergic receptor-mediated fatty acid release process, selectively coupled to the lipoxygenase route.