1. Two molecularly distinct rat P2Y receptors activated equally by adenosine-5'-triphosphate (ATP) and uridine-5'-triphosphate (UTP) (rP2Y2 and rP2Y4 receptors) were expressed in Xenopus oocytes and studied extensively to find ways to pharmacologically distinguish one from the other. 2. Both P2Y subtypes were activated fully by a number of nucleotides. Tested nucleotides were equipotent at rP2Y4 (ATP=UTP=CTP=GTP=ITP), but not at rP2Y2 (ATP=UTP>CTP>GTP>ITP). For dinucleotides (ApnA, n=2-6), rP2Y4 was only fully activated by Ap4A, which was as potent as ATP. All tested dinucleotides, except for Ap2A, fully activated rP2Y2, but none were as potent as ATP. ATP gamma S and BzATP fully activated rP2Y2, whereas ATP gamma S was a weak agonist and BzATP was inactive (as an agonist) at rP2Y4 receptors. 3. Each P2Y subtype showed different sensitivities to known P2 receptor antagonists. For rP2Y2, the potency order was suramin>>PPADS= RB-2>TNP-ATP and suramin was a competitive antagonist (pA2, 5.40). For rP2Y4, the order was RB-2>>suramin>PPADS> TNP-ATP and RB-2 was a competitive antagonist (pA2, 6.43). Also, BzATP was an antagonist at rP2Y4 receptors. 4. Extracellular acidification (from pH 8.0 to pH 5.5) enhanced the potency of ATP and UTP by 8-10-fold at rP2Y4 but did not affect agonist responses at rP2Y2 receptors. 5. Extracellular Zn2+ ions (0.1-300 microM) coapplied with ATP inhibited agonist responses at rP2Y4 but not at rP2Y2 receptors. 6. These two P2Y receptors differ significantly in terms of agonist and antagonist profiles, and the modulatory activities of extracellular H+ and Zn2+ ions. These pharmacological differences will help to distinguish between rP2Y2 and rP2Y4 receptors, in vivo.