In the present study the effects of alterations in extracellular magnesium concentration on spontaneous release of endothelium-derived relaxing factor (EDRF) and acetylcholine-, thrombin-, and calcium ionophore A23187-induced EDRF-dependent relaxation in isolated canine coronary arteries were determined. Increasing the extracellular magnesium concentration from the standard 1.2 to 2.4 mM did not alter the coronary contractile responses, while complete removal of extracellular magnesium resulted in an EDRF-dependent relaxation. The averaged percent relaxation of coronary arteries previously constricted with either prostaglandin F2 alpha, norepinephrine, or barium chloride was -75.9 +/- 4.4 (mean +/- SEM of 27 vessel segments), -43.1 +/- 4.9% (20 segments), and -25.8 +/- 6.9% (15 segments), respectively. Complete removal of extracellular magnesium also resulted in a slight but significant rightward shift of the concentration-response curves of acetylcholine- and thrombin-induced EDRF-dependent relaxation. The maximum relaxation was decreased to 86.5 +/- 1.7% and 69.5 +/- 4.6% of the control acetylcholine and thrombin relaxation, respectively. In contrast, the calcium ionophore A23187-induced EDRF-dependent relaxation was not altered following magnesium withdrawal, nor was the isoproterenol-induced endothelium-independent relaxation. These results suggest that extracellular magnesium exerts a direct inhibitory effect on the spontaneous release of EDRF and is apparently important for the expression of endothelium-dependent relaxation induced by acetylcholine and thrombin, but not calcium ionophore A23187, in canine coronary arteries.