In our efforts to identify clinically effective drugs for reversing multidrug resistance (MDR) mediated by P-glycoprotein, we tested terfenadine for anti-MDR activity because it appeared to sensitize a patient to doxorubicin and because it met structural requirements defined for this activity. Terfenadine sensitized MCF-7/ADR human breast cancer cells and L1210/VMDRC.06 murine leukemia cells to doxorubicin. At concentrations < or = 10 microM, terfenadine decreased the IC50 to doxorubicin by up to 25-fold against MCF-7/ADR cells and completely restored sensitivity to L1210/VMDRC.06 cells. The drug had no effect on the sensitive, parental cell lines and enhanced activity of other drugs affected by the MDR phenotype. Terfenadine was as potent as trans-flupenthixol, one of the most active modulators of MDR. The mechanism of action of terfenadine appeared to be due to inhibition of the function of P-glycoprotein since it augmented the accumulation of doxorubicin and inhibited the efflux of rhodamine 123 from MDR lines but had no effect on drug accumulation or efflux in sensitive cells. Terfenadine displaced azidopine from P-glycoprotein, but at concentrations higher than expected based on its overall potency. Since terfenadine is clinically available, has numerous structural derivatives available for study, and has a relatively low toxicity profile, this drug and drugs of its class should be evaluated for future clinical trials.