We have previously shown that efflux of cytotoxic drugs from multidrug resistant (MDR) cell lines can be quantitated at the single cell level using a sensitive fluorescence microscopy technique. Based on the structure of compounds which inhibited the efflux of Rhodamine-123 (Rho-123) using this methodology, we hypothesized that the antiemetic, antihistaminic agent benzquinamide (BZQ) would interfere with P-glycoprotein (P-gp) mediated drug transport and potentiate the effects of anticancer agents in MDR cell lines. We show that BZQ interferes with P-gp mediated drug efflux and increases drug accumulation in MDR cells using Rho-123 as a fluorescent probe. BZQ increases the cytotoxicity of chemotherapeutic agents to both human and hamster MDR cell lines in vitro. A slight increase in cytotoxicity to chemotherapeutic agents is also observed in the parental cell lines with BZQ. BZQ increases [3H]daunorubicin accumulation and inhibits the binding of [125I]iodoaryl azidoprazosin to the P-gp in MDR cells. BZQ is a new agent to increase the cytotoxic effects of anticancer agents in MDR cells and may ultimately prove useful as an adjunct in cancer chemotherapy.