Studies were designed to investigate effects of neutral sphingomyelinase ( N-SMase) and ceramide analogs as well as phosphorylcholine on vascular tone and Ca2+ mobilization in isolated canine cerebral arterial smooth muscle. N-SMase (0.001–0.1 U/ml) provoked a gradual but sustained vasoconstriction of arterial rings in a concentration-related manner that was endothelium independent. Incubation of denuded arterial rings in Ca2+-free medium or pretreatment with verapamil in extracellular Ca2+ resulted in a reduction of the N-SMase-evoked constriction. Exposure of arterial rings to 1,2-bis(2-aminophenoxy)ethane- N, N, N′, N′-tetraacetic acid (BAPTA)-AM did not, however, result in a reduction of N-SMase-induced constriction. Both staurosporine and bisindolymaleimide I attenuated N-SMase-induced contractions to 66% and 72% of control, respectively. N-SMase caused gradual and sustained rises in intracellular Ca2+ concentration ([Ca2+]i) in primary cultured cerebral vascular smooth muscle cells. Pretreatment of these cultured cells with nimodipine and verapamil caused a steady decline in N-SMase-induced rises in [Ca2+]i. Exposure of the cells to Ca2+-free solution reversed the [Ca2+]i-induced rise triggered by N-SMase to the resting baseline. Both C8 and C16 ceramide (10− 9–10− 6M), but not phosphorylcholine, constricted denuded canine arterial rings in a concentration-related manner and elevated [Ca2+]i. Our results suggest that the sphingomyelin-signaling pathway, via a probable release of ceramide molecules, may play an important role in regulation of cerebral arterial wall tone.