P-glycoprotein (P-gp) is a transmembrane efflux transporter which possesses many important functions in drug absorption, disposition, metabolism, and toxicity. The ultimate goal of investigating drug interactions between P-gp and drug molecules in early drug discovery is to understand the contribution of P-gp to the pharmacokinetic and pharmacodynamic properties of drug candidates and to project drug-drug interaction (DDI) potentials in humans. Understanding species differences in P-gp activities further helps the prediction of P-gp-mediated drug disposition and DDI in humans from preclinical pharmacokinetics data. The objective of the present study is to investigate the species difference in P-gp activities, via P-gp ATPase assays, using rhesus monkey Mdr1, beagle dog Mdr1, and human MDR1 expressed insect cell membranes. Twenty-one compounds with diverse chemical structures and different P-gp binding sites were chosen for the ATPase assays. P-gp ATPase binding affinities (alphaKa) and fold increases in P-gp ATPase activities (beta) of P-gp substrates were determined. Consistent with the gene and amino acid similarity, the binding affinities of test compounds to rhesus monkey P-gp were much closer to those of human P-gp than beagle dog P-gp. This is the first study which investigates the ligand affinities of P-gp from three different species. The result of this study provides an example of how to use membrane P-gp ATPase assays to evaluate interspecies P-gp differences.