In the isolated rat heart perfused with Krebs solution and prelabeled with [3H] noradrenaline, we examined the effect of prostaglandins (PG) I2, E2, 6-keto-PGF1 alpha and their precursor, arachidonic acid, on the overflow of tritium elicited by potassium (K+) and by stimulation of cardiac sympathetic nerve plexus. Prostaglandins E2, I2 and arachidonic acid but not 6-keto-PGF1 alpha reduced K+ and nerve stimulation-induced overflow of tritium. Administration of indomethacin, an inhibitor of cyclooxygenase, increased tritium overflow elicited by either K+ or by nerve stimulation. During infusion of indomethacin, the inhibitory effect of both PGE2 and PGI2 on the K+ or nerve stimulation-induced overflow of tritium remained unaltered. In contrast, the effect of arachidonic acid to reduce K+ or nerve stimulation-induced overflow of tritium was abolished by indomethacin, indicating that the fatty acid inhibits release of tritium by its conversion to a product(s) of cyclooxygenase, presumably PGI2 and PGE2. These data suggest that prostaglandins, particularly PGI2 and PGE2 synthesized in the isolated rat heart act on prejunctional sites to modulate release of the adrenergic transmitter.