Sphingolipids are important modulators of vascular tone, and we have reported that sphingosylphosphorylcholine (SPC) acts via both Rho kinase-mediated calcium sensitisation and elevation of intracellular calcium due to activation of receptor operated non-selective cation channels in rat small intrapulmonary arteries (IPA) (Thomas et al. 2005). However, the concentrations of sphingolipids required to elicit contraction in most isolated artery preparations (10-100µM) are much higher that those reported in vivo. We therefore examined whether sub-contractile concentrations of SPC affected vasoreactivity of small IPA of the rat to other stimuli. Small (300-500µm i.d.) IPA were mounted on a myograph for measurement of tension; in some experiments IPA were loaded with Fura PE-3 for measurement of intracellular calcium. Data are given as mean ± SEM, and tested for significance using either Student’s t test or ANOVA as appropriate. SPC (1µM) alone caused no contraction and either no or a very small increase in intracellular calcium. However, the concentration-response relationships for depolarising potassium concentrations and prostaglandin F2α (PGF) were significantly shifted to the left (EC₅₀ potassium, control: 41 ± 4mM, SPC: 31 ± 2mM, n=9, p<0.05; PGF control: 18 ± 7µM, SPC: 9 ± 1µM, n=11, p<0.05). In separate experiments, pre-treatment with 1 µM SPC potentiated constriction induced by 23mM potassium by 460 ± 57% after 30min (n=11, p<0.01). This potentiation was not significantly affected by removal of the endothelium, inhibition of Rho kinase with Y-27632 (10µM), or blockade of non-selective cation channels with 10µM lanthanum or 75µM 2-aminophenylborate (2-APB) (n=5-6). Following inhibition with the PKC inhibitor Ro 31-8220 (3µM), however, the potentiation was reduced and was no longer significant (160 ± 54%, n=5). Addition of 1µM SPC to PGF-preconstricted IPA caused a smaller but significant increase in tension (40 ± 7%, n=10, p<0.01), which was also resistant to Y-27632 (n=6) but suppressed by Ro 31-8220 (6 ± 4%, n=6, p<0.001). The potentiation was halved by diltiazem (10μM; 21 ± 3%, n=4, p<0.05), but abolished by 2-APB (4 ± 4%, n=4, p<0.001). Further experiments showed that 1µM SPC substantially increased the rise in intracellular calcium induced by either ~23mM potassium or 100nM U-46619 (a TP agonist), and that these increases were suppressed by PKC inhibition (n=4-5). We propose that sub-contractile concentrations of SPC increase vasoreactivity in IPA via a PKC-dependent mechanism that enhances calcium entry via both voltage dependent L-type channels, and non-selective, receptor operated channels. This may have implications for the physiological relevance of SPC.