In the absence of selective antagonists, pharmacological characterization of P2Y receptor subtypes has relied heavily upon their distinct agonist profiles. 2-methylthioADP (2-MeSADP) is a selective agonist for the P2Y(1) receptor. The agonist action of 2-MeSATP at the P2Y(1) receptor has recently been questioned. The effects of both 2-MeSADP and 2-MeSATP have been studied on rat hepatocytes injected with the bioluminescent Ca(2+) indicator, aequorin. Single hepatocytes generate series of repetitive transients in cytosolic free calcium concentration ([Ca(2+)](i)) when stimulated with agonists acting through the phosphoinositide signalling pathway. The transients induced by 2-MeSADP and 2-MeSATP in the same cell were indistinguishable, indicating that they act at a common receptor. In contrast the transients evoked by ATP and UTP had very different profiles. Treatment of 2-MeSATP with an ATP-regenerating system to remove contaminating 2-MeSADP did not abolish its agonist activity. Application of the P2Y(1) antagonist, adenosine-3'-phosphate-5'-phosphate (A3P5P) inhibited the transients induced by both 2-MeSADP and 2-MeSATP. In contrast the transients induced by ATP and UTP were enhanced by the addition of A3P5P. These results indicate that both 2-MeSADP and 2-MeSATP are agonists at the rat hepatocyte P2Y(1) receptor.