The human P2Y13 receptor is a new receptor characterized by coupling to Gi, responsiveness to adenine di-phospho-nucleotides and blockade by the P2Y antagonist AR-C69931MX. The mouse P2Y13 ortholog has also been reported. Here we report, for the first time, the cloning of rat P2Y13 receptor, its pharmacological analysis and tissue distribution. Rat P2Y13 is 79% and 87% identical to human and mouse P2Y13 receptors, respectively. Expression of rP2Y13 receptor in 1321N1 cells induced the appearance of responses to the typical P2Y13 receptor agonists ADP and 2MeSADP, as detected by stimulation of [35S]GTPγS binding. Agonist activities were higher in cells transfected with rP2Y13 receptor in the presence of the Gα16 subunit; in all cases agonist effects were abolished by pertussis toxin pre-treatment. At variance from both human and mouse receptors, ADP was more potent than 2MeSADP. Other nucleotides and sugar-nucleotides were ineffective. Both in the absence and presence of Gα16, activation of rP2Y13 receptor by ADP and 2MeSADP was completely inhibited by nM concentrations of AR-C69931MX. In contrast, no inhibition of rP2Y13 receptor was induced by the selective P2Y1 receptor antagonist MRS2179. rP2Y13 receptor showed highest expression levels in spleen, followed by liver and brain (with particularly high levels in cortex and striatum as reported in man), suggesting important roles in the nervous and immune systems. Expression levels comparable to those of the other cloned P2Y receptors were found in primary rat astrocytes, indicating a possible role in reactive astrogliosis. Hence, rat P2Y13 receptor displays several similarities but also interesting differences with its human and mouse orthologs, that will have to be taken into account when characterizing the pathophysiological roles of this receptor in the rat animal models.