Considerable uncertainty surrounds the stoichiometry of coupling of ATP hydrolysis to drug pumping by P-glycoprotein, the multidrug transporter. To estimate relative turnovers for pumping of the drug vinblastine and ATP hydrolysis, we began by measuring the number of P-glycoprotein molecules on the surface of murine NIH3T3 cells expressing the human MDR1 gene. Fluorescence of cells treated with monoclonal antibody UIC2 was determined as a function of (i) amount of antibody at a fixed number of cells and (ii) increasing cell number at constant antibody. The two together gives 1.95 x 10(6) P-glycoprotein molecules/cell. Initial uptake rates of vinblastine +/- verapamil measure the ability of P-glycoprotein to extract vinblastine from the plasma membrane before it enters the cell. As a function of [vinblastine] at 37 degrees C, they give the maximum rate of this component of outward pumping as 2.1 x 10(6) molecules s-1 cell-1 or a turnover number of 1.1 s-1. Initial rates of one-way efflux as a function of [vinblastine] at 25 degrees C +/- glucose give the maximum rate of this component of pumping as 0.59 x 10(6) molecules s-1 cell-1. The ratio of ATPase activity of P-glycoprotein at 37 and 25 degrees C is 4.6. Appropriating this ratio for pumping, maximum one-way efflux at 37 degrees C is 4.6 x 0.59 = 2.7 x 10(6) molecules s-1 cell-1, a turnover number of 1.4 s-1. The vinblastine-stimulated ATPase activity of P-glycoprotein has a turnover number of 3.5 s-1 at 37 degrees C, giving 2.8 molecules of ATP hydrolyzed for every vinblastine molecule transported in a particular direction. These calculations involve several approximations, but turnover numbers for pumping of vinblastine and for vinblastine-stimulated ATP hydrolysis are comparable. Thus, ATP hydrolysis is probably directly linked to drug transport by P-glycoprotein.