We have investigated the involvement of intracellular pH (pH(i)) in the regulation of P-glycoprotein (P-gp) in K562/DOX cells. The selective Na(+)/H(+) exchanger1 (NHE1) inhibitor cariporide and the "high K(+)" buffer were used to induce the sustained intracellular acidification of the K562/DOX cells that exhibited more alkaline pH(i) than the K562 cells. The acidification resulted in the decreased P-gp activity with increased Rhodamine 123 (Rh123) accumulation in K562/DOX cells, which could be blocked by the P-gp inhibitor verapamil. Moreover, the acidification decreased MDR1 mRNA and P-gp expression, and promoted the accumulation and distribution of doxorubicin into the cell nucleus. Interestingly, these processes were all pH(i) and time-dependent. Furthermore, the change of the P-gp expression was reversible with the pH(i) recovery. These data indicate that the tumor multidrug resistance (MDR) mediated by P-gp could be reversed by sustained intracellular acidification through down-regulating the P-gp expression and activity, and there is a regulative link between the pH(i) and P-gp in K562/DOX cells.