The possibility that simple lipophilic cations such as tetraphenylphosphonium (TPA+), triphenylmethylphosphonium (TPMP+), and diphenyldimethylphosphonium (DDP+) are substrates for the multidrug-resistance transport protein, P-glycoprotein, was tested. Hamster cells transfected with and overexpressing mouse mdr1 or mouse mdr3 exhibit high levels of resistance to TPP+ and TPA+ (20-fold) and somewhat lower levels of resistance to TPMP+ and DDP+ (3-12-fold). Transfected cell clones expressing mdr1 or mdr3 mutants with decreased activity against drugs of the MDR spectrum (e.g., Vinca alkaloids and anthracyclines) also show reduced resistance to lipophilic cations. Studies with radiolabeled TPP+ and TPA+ demonstrate that increased resistance to cytotoxic concentrations of these lipophilic cations is correlated quantitatively with a decrease in intracellular accumulation in mdr1- and mdr3-transfected cells. This decreased intracellular accumulation is shown to be strictly dependent on intact intracellular nucleotide triphosphate pools and is reversed by verapamil, a known competitive inhibitor of P-glycoprotein. Taken together, these results demonstrate that lipophilic cations are a new class of substrates for P-glycoprotein and can be used to study its mechanism of action in homologous and heterologous systems.