The role of cyclic nucleotides in the dilation of rat aortic rings induced by isoprenaline was studied by measuring changes in isometric tension and in cyclic nucleotide levels in rings preconstricted with phenylephrine or U46619. Isoprenaline produced significantly greater relaxation in endothelium-containing than in endothelium-denuded rings. Relaxation of endothelium-containing rings to isoprenaline was markedly inhibited by pretreating the rings with methylene blue (30 microM) or haemoglobin (10 microM). Basal cyclic-AMP levels were not affected by the presence of endothelium in the rings, but basal cyclic-GMP levels were 5-6-fold higher in endothelium-containing rings. Isoprenaline (1 microM) elevated levels of cyclic-AMP to an extent which was not affected by the presence of endothelium. In contrast, isoprenaline had no effect on cyclic-GMP levels in rings with or without endothelium. Pretreatment of de-endothelialized rings with sodium nitroprusside or 8-bromo-cyclic-GMP, in concentrations that by themselves caused minor alterations in vascular tone, markedly amplified the relaxant responses to isoprenaline and mimicked the relaxant responses of endothelium-containing rings to isoprenaline. These data indicate that isoprenaline does not stimulate release of endothelium-derived relaxing factor, but suggest a major synergistic interaction between cyclic-GMP-mediated and cyclic-AMP-mediated relaxation in rat aorta.