Acidification of the extracellular fluid modulates neurotransmission and ischemic injury in brain. The purpose of the present study was to investigate the effects of purine and pyrimidine transmitters on acid efflux from brain astrocytes. Using RT‐PCR, we detected transcripts for the following nucleotide receptors in rat primary astrocyte cultures: P2X1, P2X2, P2X3, P2X4, P2X6, P2X7, P2Y1, P2Y2, P2Y4, and P2Y6. Adenosine 5′‐triphosphate (ATP) was found to rapidly induce a biphasic increase in acid efflux, monitored by microphysiometry, consisting of an initial transient and a sustained plateau. Compared with ATP, the P2Y agonist uridine 5′‐triphosphate (UTP) induced a much smaller initial response but an equal plateau. The poorly hydrolyzable ATP analogue ATPγS caused the same initial response as did ATP, but a much smaller plateau, suggesting that the latter phase was due to extracellular degradation of nucleotides. The P2 receptor antagonist, suramin, blocked stimulation of acid efflux by ATP. Removal of extracellular glucose or elevation of extracellular K⁺ decreased the basal rate of acid efflux but not the stimulation induced by ATP. Inhibition of Na⁺/H⁺ exchange by cariporide suppressed the initial phase of ATP‐stimulated acid efflux. The intracellular Ca²⁺ chelator bisaminophenoxyethane‐ tetraacetic acid (BAPTA) lowered basal acid efflux and abolished the initial phase of the response to ATP. In conclusion, ATP acts through P2 nucleotide receptors on astrocytes to stimulate the Ca²⁺‐dependent efflux of protons, mediated in part by activation of Na⁺/H⁺ exchange. The resulting acidification of the extracellular fluid may serve as an intercellular signal in brain. © 2004 Wiley‐Liss, Inc.