The effect of ouabain on exocytotic and nonexocytotic norepinephrine release was investigated in perfused rat and guinea pig hearts. The overflow of endogenous norepinephrine and its neuronal metabolite 3,4-dihydroxyphenylethyleneglycol (DOPEG) was determined by high-pressure liquid chromatography. DOPEG served as the indicator of free axoplasmic norepinephrine concentrations. The overflow of the norepinephrine cotransmitter neuropeptide Y (NPY) was determined by radioimmunoassay and NPY was used as marker for exocytotic release. Electrical stimulation of the left stellate ganglion resulted in exocytotic norepinephrine release in rat and guinea pig hearts. Ouabain caused an increase in stimulation-induced norepinephrine overflow from rat and guinea pig hearts by 40%. However, overflow of NPY was decreased by 40%, indicating a reduced exocytosis rate. Ouabain increased both norepinephrine and NPY overflow, suggesting enhancement of exocytosis, when neuronal catecholamine uptake (uptake1) was blocked by desipramine or when presynaptic alpha 2-adrenoceptors were inhibited by yohimbine. The results demonstrate an interaction of ouabain with both calcium-dependent exocytosis and uptake1 of norepinephrine. Under calcium-free conditions, ouabain or potassium-free perfusate resulted in norepinephrine release from hearts when the axoplasmic norepinephrine concentration was elevated by the reserpinelike agent Ro 4-1284. This release was independent from neural activity, not accompanied by NPY overflow, and suppressed by the uptake1 blocker desipramine. These findings are in keeping with carrier-mediated nonexocytotic norepinephrine release that is caused by reversal of the transport direction of the uptake1 carrier. During myocardial ischemia nonexocytotic norepinephrine release was accelerated and enhanced by inhibition of Na+,K(+)-ATPase before ischemia. This study demonstrates the potential of digitalis glycosides to interact both with transmitter exocytosis and with the neuronal catecholamine transport system by Na+,K(+)-ATPase inhibition. Interaction with the catecholamine transport system involves both inhibition of norepinephrine inward transport and induction of norepinephrine outward transport, resulting in nonexocytotic norepinephrine release.