Many of the pharmacological effects of Delta(9)-tetrahydrocannabinol are mediated through CB(1) and CB(2) cannabinoid receptors. However, with the discovery of endogenous cannabinoids, some discrepancies have arisen. Furthermore, unlike the CB(1) receptor, the sequences of the mouse and human CB(2) receptor are divergent, raising the possibility of species specificity. The gene for the rat CB(2) receptor was cloned, expressed, and its properties compared with those of mouse and human CB(2) receptors. Sequence analysis of the coding region of the rat CB(2) genomic clone indicates 90% nucleic acid identity (93% amino acid identity) between rat and mouse and 81% nucleic acid identity (81% amino acid identity) between rat and human. The rat CB(2) receptor was stably expressed in human embryonic kidney-293 cells to examine its pharmacology. The rat CB(2) showed low affinity for anandamide, an endogenous ligand shown to act at the CB(1) receptor. In contrast, high-affinity binding for SR144528 (CB(2)-selective antagonist) as well as several cannabinoid receptor agonists was observed. Coupling to adenylate cyclase was observed. Aspects of the pharmacology of palmitoylethanolamide were also examined. It bound to CB(1) and CB(2) receptors with low affinity and stimulated GTPgammaS binding in the cerebellum and CB(2)-expressing cell lines with low potency. The data in this study suggest that the discrepancies in affinities between rat and human may represent species differences. The rat CB(2) receptor genomic clone will be a useful tool for studying the function and regulation of CB(2) in rats.