Maintenance and regulation of intracellular pH (pHi) was studied in wild-type Ehrlich ascites tumor cells (EHR2) and five progressively daunorubicin-resistant, P-glycoprotein (P-gp)-expressing strains, the maximally resistant of which is EHR2/1.3. Steady-state pHi was similar in cells expressing different amounts of P-gp, in the absence and presence of glucose. In EHR2/1.3, glucose-induced acidification was reduced, and proton efflux was increased, compared to the wild-type EHR2, differences which were not caused by increased activity of a Na+/H+ exchanger in the resistant cells. Comparing all six cell lines, no evidence was found for a correlation between the amount of P-gp in the membrane and pHi regulation, which was also unaffected by P-gp modulators. However, a correlation was seen between relative resistance/daunorubicin accumulation and acid extrusion rate, which is likely to be due to aspects of development of drug resistance other than P-gp.