Many physiological and pathophysiological situations generate a significant increase in extracellular K⁺ concentration. This is known to influence a number of membrane conductances and exchangers, whereas direct effects of K⁺ on the activation of G protein-coupled receptors have not been reported. We now show that Ca²⁺ release evoked by P2Y1 receptors expressed in 1321-N1 astrocytoma cells is markedly potentiated by small increases in external K⁺ concentration. This effect was blocked by the phospholipase-C inhibitor U-73122 (1-[6-[[17 beta]-methoxyestra-1,3,5(10)-trien-17-yl]amino]hexyl]-1H-pyrrole-2,5-dione), but not by its analog U-73343 (1-[6-[[17 beta]-3-methoxyestra-1,3,5(10)-trien-17-yl]amino]hexyl]-2,5-pyrrolidinedione), and not by nifedipine, Ni²⁺, Cd²⁺, or Gd³⁺. Thus, K⁺ enhances d-myo-inositol 1,4,5-trisphosphate-dependent Ca²⁺ release without a requirement for Ca²⁺ influx. The cation dependence of this effect displayed the order K⁺ > Rb⁺ > N-methyl-D-glucamine⁺, and Cs⁺ and choline⁺ were ineffective. The potentiation by K⁺ is half-maximal at an increase of 2.6 mM (total K⁺ of 7.6 mM). K⁺ caused a reduction in EC50 (2.7-fold for a 29 mM increase) without a change of slope; thus, the greatest effect was observed at near-threshold agonist levels. The response to K⁺ can be explained in part by depolarization-dependent potentiation of P2Y1 receptors [J Physiol (Lond) 555:61-70, 2004]. However, electrophysiological recordings of 1321-N1 cells and megakaryocytes demonstrated that K⁺ also amplifies ADP-evoked Ca²⁺ responses independently of changes in membrane potential. Elevated K⁺ also amplified endogenous UTP-dependent Ca²⁺ responses in human embryonic kidney 293 cells, suggesting that other P2Y receptors are K(⁺)-dependent. P2Y receptors display a widespread tissue distribution; therefore, their modulation by small changes in extracellular K+ may represent a novel means of autocrine and paracrine regulation of cellular activity.