1. The involvement of prostacyclin (PGI2) in the vasodilator responses to acetylcholine (ACh), A23187 and bradykinin (Bk) has been investigated in guinea-pig, isolated, Krebs-perfused hearts. 2. ACh (0.01-10 nmol), A23187 (0.1-1.0 nmol) and Bk (0.3-10 pmol) each elicited dose-related and shortlasting (approximately 2 min) reductions in perfusion pressure. Larger maximal responses were obtained in preparations with coronary vascular tone elevated by platelet-activating factor (100 pmol) than in preparations at basal perfusion pressure. 3. Bk and A23187 elicited dose-related increases in the generation of PGI2 as measured by its chemically-stable breakdown product, 6-oxo-PGF1 alpha. Indomethacin (2.8 microM) prevented both basal and the stimulated generation of 6-oxo-PGF1 alpha, whereas the magnitudes of the vasodilator responses were unaffected. 4. Attempts to identify the release of vasodilator materials by on-line superfusion bioassay of cardiac effluent were unsuccessful, indicating a possible role for a labile vasodilator such as endothelium-dependent relaxing factor (EDRF). In addition, the inhibitors of EDRF action/production, mepacrine (3 microM) or diethylcarbamazine (300 microM), attenuated vasodilator responses to ACh without altering those to the endothelium-independent vasodilator, verapamil (1 nmol). 5. Haemoglobin (10 microM) reduced vasodilator responses to ACh, Bk and verapamil and abolished those induced by A23187. Inhibition of the endothelium-independent vasodilator, verapamil, was significantly less than that for the other compounds. 6. The present data indicate the existence of an indomethacin-resistant vasodilator mechanism in the coronary microcirculation in response to ACh, A23187 and Bk. EDRF is a candidate for mediating these responses; however, a direct vasodilator action of these substances cannot be excluded.