Abstract—Sphingosylphosphorylcholine (SPC) is a powerful vasoconstrictor, but in vitro its EC₅₀ is 100-fold more than plasma concentrations. We examined whether subcontractile concentrations of SPC (1 µmol/L) modulated vasoreactivity of rat intrapulmonary arteries using myography and measurement of intracellular [Ca²⁺]. SPC (1 µmol/L) had no effect on force or intracellular [Ca²⁺] on its own, but dramatically potentiated constrictions induced by 25 mmol/L [K⁺], such that at 40 minutes, force and intracellular [Ca²⁺] (Fura PE3 340/380 ratio) were increased by 429±96% and 134±26%, respectively. The potentiation was stereospecific, apparent at concentrations >100 nmol/L of SPC, and independent of the endothelium, 2-aminoethoxydiphenylborane–sensitive Ca²⁺ entry, and Rho kinase. It was abolished by the phospholipase C inhibitor U73122, the broad spectrum protein kinase C (PKC) inhibitor Ro31-8220, and the PKC inhibitor rottlerin, but not by Gö6976, which is ineffective against PKC. The potentiation could be attributed to enhancement of Ca²⁺ entry. SPC also potentiated the responses to prostaglandin F₂ and U436619, which activate a 2-aminoethoxydiphenylborane sensitive nonselective cation channel in intrapulmonary arteries. In this case, potentiation was partially inhibited by diltiazem but abolished by 2-aminoethoxydiphenylborane, Ro31-8220, and rottlerin. SPC (1 µmol/L) caused translocation of PKC to the perinuclear region and cytoskeleton of cultured intrapulmonary artery smooth muscle cells. We present the novel finding that low, subcontractile concentrations of SPC potentiate Ca²⁺ entry in intrapulmonary arteries through both voltage-dependent and independent pathways via a receptor-dependent mechanism involving PKC. This has implications for the physiological role of SPC, especially in cardiovascular disease, where SPC is reported to be elevated.