The sphingomyelin pathway is now recognized as an important signal transduction system regulating various cellular functions, in which activation of a neutral sphingomyelinase induced by various extracellular stimulants results in selective degradation of sphingomyelin, yielding bioactive lipid intermediates, ceramides and phosphorylcholine. In the present study, our emphasis has been to examine the effects of exogenous Mg(2+)-dependent neutral sphingomyelinase, in physiological and pathophysiological magnesium concentrations, on phenylephrine-induced vasomotor tone and on intracellular free Ca(2+)([Ca(2+)](i)) mobilization in vitro. Neutral sphingomyelinase (0.001-0.1 U/ml), alone, did not elicit any significant changes in either basal tension or resting levels of [Ca(2+)](i) in rat aortic smooth muscle; similar results were obtained with phosphorylcholine. However, neutral sphingomyelinase (0.001-0.1 U/ml) and C(2)-ceramide or ceramide-1-phosphate, but not phosphorylcholine, attenuated phenylephrine-induced contractions, in isolated rat aortic rings, in a concentration-related manner. The addition of extracellular magnesium in different concentrations (0, 0.3, 1.2, 2.4 mM) modulated the neutral sphingomyelinase-vasorelaxant action in a concentration-dependent manner. Neutral sphingomyelinase-evoked relaxation was only partially endothelium-dependent. Nitric oxide synthase inhibitors, N(G)-nitro-L-arginine (L-NNA) and L-N(G)-monomethyl-arginine (L-NMMA), an inhibitor of prostanoid synthesis (indomethacin), and pharmacologic amine antagonists, such as atropine, diphenhydramine, cimetidine, propranolol, and methysergide as well as an opiate antagonist, naloxone, all failed to attenuate or interfere with the vasorelaxant responses of neutral sphingomyelinase. Three different inhibitors of protein kinase C (i.e., staurosporine, 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H-7) or bisindolylmaleimide I), when used over a wide concentration range, also failed to interfere with the neutral sphingomyelinase-induced relaxations. Neutral sphingomyelinase inhibited the elevations in [Ca(2+)](i) in cultured rat aortic smooth muscle cells caused by phenylephrine. Our results suggest that a Mg(2+)-dependent sphingomyelin signaling pathway may play an important regulatory role in arterial wall tone.