Activation of IK(ACh) is the major effect of the vagal neutrotransmitter acetylcholine in the heart. We report that both lysosphingomyelin (D-erythro-sphingosyl-phosphorylcholine; SPC) and sphingosine 1-phosphate (SPP) activate IK(ACh) in guinea pig atrial myocytes through the same receptor with an EC50 of 1.5 and 1.2 nM, respectively. Pertussis toxin abolished the activation of IK(ACh) by either lipid. The putative receptor showed an exquisite stereoselectivity for the naturally occurring D-erythro-(2S,3R)-SPC stereoisomer, the structure of which was confirmed by mass spectroscopy and NMR. These lipids caused complete homologous and heterologous desensitization with each other but not with ACh, indicating that both act on the same receptor. This receptor displays a distinct structure-activity relationship: it requires an unsubstituted amino group because N-acetyl-SPC, lysophosphatidic acid and lysophosphatidylcholine were inactive. Because SPP and SPC are naturally occurring products of membrane lipid metabolism, it appears that these compounds might be important extracellular mediators acting on a family of bona fide G protein-coupled receptors. Expression of these receptors in the heart raises the possibility that sphingolipids may be a part of the physiological and/or pathophysiological regulation of the heart. Based on their ligand selectivity we propose a classification of the sphingolipid receptors.