Extracellular acidification rates (ECARs) in response to eight different drugs activating or inhibiting the ATPase of P-glycoprotein (Pgp) were measured in real time by means of a Cytosensor microphysiometer in MDR1-transfected and corresponding wild-type cell lines, i.e., pig kidney cells (LLC-MDR1 and LLC-PK1) and mouse embryo fibroblasts (NIH-MDR-G185 and NIH3T3). The ECARs showed a bell-shaped dependence on drug concentration (log scale) in transfected cells but were negligibly small in wild-type cells. The activation profiles (ECARs vs concentration) were analyzed in terms of a model assuming activation of Pgp-ATPase with one and inhibition with two drug molecules bound. The kinetic constants [concentration of half-maximum activation (inhibition), K(i), and the maximum (minimum) transporter activity, V(i)] were in qualitative and quantitative agreement with those determined earlier for Pgp-ATPase activation monitored by phosphate release in inside-out cellular vesicles and in purified reconstituted systems, respectively. Furthermore, the ECARs correlated with the expression level of Pgp in the two different cell lines and were reduced in a concentration-dependent manner by cyclosporin A, a potent inhibitor of the Pgp-ATPase. In contrast, treatment of cells with inhibitors of the Na(+)/H(+) or the Cl(-)/HCO(3)(-) exchanger did not reduce the ECARs. The micro-pH measurements provide for the first time direct evidence for a tight coupling between the rate of extracellular proton extrusion and intracellular phosphate release upon Pgp-ATPase activation. They support a Pgp-mediated transport of protons from the site of ATP hydrolysis to the cell surface. Measurement of the ECARs could thus constitute a new method to conveniently analyze the kinetics of Pgp-ATPase activation in living cells.