The effect of extracellular ATP (10–100 μm) on intracellular Ca2+ concentration ([Ca2+]i) and firing rate has been studied in single pacemaker cells isolated from the sinus venosus of cane toads. In spontaneously firing cells, ATP initially increased peak [Ca2+]i by 43 ± 5 %, increased diastolic [Ca2+]i by 20 + 3 % and increased the firing rate by 58 ± 8 %. These early effects were followed by a late phase in which both the peak [Ca2+]i and the firing rate declined. Adenosine, and UTP (respectively, P1- and P2Y2,4,6-selective agonists) caused no significant change in [Ca2+]i or firing rate, while αβ-methylene ATP (a P2X1,3 agonist) caused a small increase in firing rate but no changes in [Ca2+]i. In contrast the P2Y1-selective agonist 2-MesADP (1 μm) mimicked the biphasic effects of ATP and these effects were inhibited by the purinoceptor antagonists suramin and PPADS and by the P2Y1-selective antagonist MRS 2179. Immunohistochemistry established that P2Y1 purinoceptors were present on the cell surface. Western blotting analysis demonstrated that the P2Y1 antibody recognised a 57 kDa protein. After sarcoplasmic reticulum Ca2+ release was prevented with caffeine or ryanodine, ATP no longer had any effect on [Ca2+]i or firing rate. Furthermore, the SR Ca2+ store content was decreased during the late phase of 2-MesADP application. The effect of ATP was coupled to phospholipase C (PLC) activity because the PLC inhibitor U-73122 eliminated the effects of ATP. Our study shows that in toad pacemaker cells, the biphasic effects of ATP on pacemaker activity are mainly through P2Y1 purinoceptors, which are able to modulate Ca2+ release from the SR Ca2+ store.