The present study used a stationary microperfusion technique to investigate in vivo the effect of P2Y(1) receptor activation on bicarbonate reabsorption in the rat proximal tubule. Proximal tubules were perfused with a bicarbonate Ringer solution before flow was stopped by means of an oil block. The recovery of lumen pH from the initial value ( pH 8.0) to stationary values ( pH similar to6.7) was recorded by a H+-sensitive microelectrode inserted downstream of the perfusion pipette and oil block. The stationary pH value and the t(1/2) of pH recovery were used to calculate bicarbonate reabsorption (J(HCO3)). Both EIPA and bafilomycin A1 caused significant reductions in proximal tubule J(HCO3), consistent with the established contributions of Na/H exchange and H+-ATPase to proximal tubule HCO3 reabsorption. The nucleotides ADP and, to a lesser extent, ATP reduced J(HCO3) but AMP and UTP were without effect. 2MeSADP, a highly selective agonist of the P2Y(1) receptor, reduced J(HCO3) in a dose-dependent manner. MRS-2179, a P2Y(1) receptor-specific antagonist, abolished the effect of 2MeSADP, whereas theophylline, an antagonist of adenosine (P1) receptors, did not. The inhibitory action of 2MeSADP was blocked by inhibition of protein kinase C and reduced by inhibition of protein kinase A. The effects of EIPA and 2MeSADP were not additive. The data provide functional evidence for P2Y(1) receptors in the apical membrane of the rat proximal tubule: receptor activation impairs acidification in this nephron segment.