To our knowledge, this is the first study to investigate the modification of P-glycoprotein functionality in living resistant cells after photolabeling. For this purpose, four new photoactive verapamil analogues were synthesized. These compounds have the same efficacy as verapamil to increase pirarubicin (pira) incorporation into living multidrug resistant (MDR) K562 cells and to sensitize them to the cytotoxic effect of this anthracycline derivative, indicating that they act as typical MDR modifiers in MDR cells. These compounds were used to photolabel P-glycoprotein (P-gp) in living resistant cells. Irradiation did not result in photodamage to cells, and P-gp functionality was verified by the ability of living cells to incorporate pira. The irradiation of resistant cells, 10(6)/mL, in the presence of a verapamil analogue at concentrations equal to or higher than 3 microM yielded 70% inhibition of P-gp functionality. Our data provide the first evidence that the binding of a verapamil analogue to P-gp is not sufficient to completely inhibit the efflux of this anthracycline. The cells were, subsequently, cultured for several days. Resistance was progressively recovered with time, with the treated cells being just as resistant as before photolabeling after 6 days.