In the present study, we determined the agonist specificity and the signalling mechanisms of a putative sphingosine 1-phosphate (S1P) receptor, AGR16. In CHO cells transiently transfected with an AGR16 expression vector, but not in cells transfected with an empty vector, the addition of a low concentration of S1P (1 nM) caused an increase in the intracellular free Ca2+ concentration ([Ca2+]i) by mobilization of Ca2+ from both intra- and extra-cellular pools. To determine the spectrum of agonists for AGR16, we employed K562 cells, which in the naive state do not respond at all to either S1P or structurally related lipids with an increase in [Ca2+]i. In K562 cells stably expressing AGR16, S1P and sphingosylphosphorylcholine (SPC) dose-dependently increased [Ca2+]i with half-maximal values of 3 nM and 100 nM respectively. In CHO cells stably expressing AGR16 (CHO-AGR16), but not in parental CHO cells, we observed specific binding of [32P]S1P, which was displaced by unlabelled S1P and SPC. In CHO-AGR16 cells, but not in parental CHO cells, S1P stimulated the production of inositol phosphates and Ca2+ mobilization which was only 30% inhibited by pertussis toxin (PTX), different from the case of the recently identified S1P receptor EDG1. Also in CHO-AGR16 cells, but not in CHO cells, S1P at higher concentrations activated mitogen-activated protein kinase (MAPK) in a PTX-sensitive and Ras-dependent manner. S1P also induced the activation of two stress-activated MAPKs, c-Jun N-terminal kinase and p38, in a manner that was totally insensitive to PTX. In CHO-AGR16 cells, S1P induced stress-fibre formation, with an increase in myosin light chain phosphorylation, in a PTX-insensitive and Rho-dependent manner. S1P also induced an increase in the cellular cAMP content in CHO-AGR16 cells, which contrasts sharply with the case of EDG1. These results establish that the S1P receptor AGR16 is coupled via both PTX-sensitive and -insensitive G-proteins to multiple effector pathways.