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5-HT1Areceptors couple to many signaling pathways in CHO-K1 cells through pertussis toxin-sensitive G proteins. The purpose of this study was to determine which members of the Gi/o/zfamily mediate 5-HT1Areceptor-activated Na+/H+exchange as measured by microphysiometry of cell monolayers. The method was extensively validated, showing that proton efflux was sodium-dependent, inhibited by amiloride analogs, and activated by growth factors, phorbol ester, calcium ionophore, and hypertonic stress. 5-HT and the specific agonist (±)-8-hydroxy-2-(di-N-propylamino)tetralin hydrobromide rapidly stimulated proton efflux that was blocked by a specific receptor antagonist, amiloride analogs or pertussis toxin. The activation by 5-HT depended upon extracellular sodium and could be demonstrated under conditions of imposed intracellular acid load, as well as in the presence and absence of glycolytic substrate. Acceleration of proton efflux was not inhibited by sequestration of G protein βγ-subunits, a maneuver that blocked 5-HT1Areceptor activation of mitogen-activated protein kinase. Transfection of Gzαand pertussis toxin-resistant mutants of Goαand Giα1did not reverse the blockade induced by pertussis toxin. In contrast, pertussis toxin-resistant mutants of Giα2and Giα3“rescued” the ability of 5-HT to increase proton efflux after pertussis toxin treatment. These experiments demonstrate clearly that Giα2and Giα3can specifically mediate rapid agonist-induced acceleration of Na+/H+exchange.