Abstract Mode and site of release of ATP evoked by isoprenaline were evaluated in the electrically driven left atrial segment of guinea pig. The peak release of ATP 5 min after 1 μM isoprenaline was inhibited by 1 μM propranolol and 1 μM butoxamine, but not by 1 μM atenolol, showing that the ATP release is due to simulation of the presynaptic β 2-adrenoceptor by isoprenaline. The maximum ATP release was markedly reduced by Ca 2+/calmodulin antagonists, W-7 and trifluoperazine, and by a mitotic inhibitor, vinblastine. Further, the release was similarly inhibited by myosin light chain kinase inhibitors, ML-7 and wortmannin. Nifedipine, a Ca 2+-channel blocker, decreased the release of ATP evoked by isoprenaline. By contrast, Bay K 8644, a Ca 2+-channel opener, tended to enhance the ATP release. These findings suggest that isoprenaline produces ATP release from adrenergic nerve terminals of atrium, implying that ATP serves as a co-transmitter.