The characteristics of ATP release evoked by forskolin and ouabain from atrial segments of guinea-pig were evaluated under electrical stimulation. Forskolin (1 μM) produced a massive release of ATP together with a positive inotropic response. Both 30 μM W-7 (N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide HCl), a calmodulin antagonist, and 30 μM vinblastine, a mitotic inhibitor, markedly inhibited the evoked release of ATP without affecting the evoked contraction. However, 100 μMN -ethylmaleimide abolished completely the basal and drug-evoked ATP release and further the evoked contraction. Both the ATP release and contraction evoked by ouabain (3 μM) were similarly affected by W-7, vinblastine and n-ethylmaleimide. The release of ATP, but not the contraction, evoked by forskolin was strongly suppressed by 10 μM okadaic acid, a protein phosphatase inhibitor. The suppression by okadaic acid of the evoked release was thoroughly antagonized in the presence of 0.01 μM PMA (phorbol 12-myristate 13-acetate), but not 10 μM H-7 (1-(5-isoquinolinesulfonyl)-2-methylpiperazine). These results suggest that forskolin, like ouabain, may dominantly cause the neuronal release of ATP from cardiac adrenergic nerves, although the possible participation of release from muscular sources cannot be ignored.