P-glycoprotein (P-gp) plays an important role in the pharmacokinetics of drugs. There is little information on the species differences in P-gp-mediated drug transport activity. The purpose of the present study was to clarify the differences in the kinetic parameters and the existence of species differences in the P-gp-mediated drug transport activity using seven multidrug resistence1 (MDR1) transfected cell lines, in which the cDNA was from human, monkey, canine, rat (MDR1a and MDR1b), and mouse (mdr1a and mdr1b). The transcellular transport of diltiazem, cyclosporin A, and dexamethasone across monolayers of MDR1 transfected cells. The apparent K(m) values of diltiazem exhibited approximately 16.5-fold differences among the seven cell lines. Concerning the diltiazem transport, the V(max)/K(m) value of human P-gp corrected by the P-gp expression level was similar to that of monkey P-gp, but was 5.6-fold higher than that of canine P-gp. On the other hand, the corrected V(max)/K(m) value of human P-gp for cyclosporin A transport was 3.8-fold higher than that of monkey P-gp. The present study would be valuable to evaluate the P-gp function of various animals in the same experimental condition. It was clarified that the species differences in P-gp-mediated drug transport activity evaluated by the corrected V(max)/K(m) value differed according to the substrate.