Verapamil sensitizes multidrug-resistant cell lines to various heterocyclic anticancer drugs by inhibition of energy-dependent release of drug, presumably by interaction with membrane glycoproteins involved in drug efflux. This work assessed verapamil sensitization of human multidrug-resistant lymphocytic and myeloid leukemic cell lines (CEM/VLB100, HL-60/AR) to vincristine during exposures of short duration (4 h). When cells were transferred to drug-free medium immediately after simultaneous 4-h exposures to vincristine and verapamil, the antiproliferative activity of vincristine was not altered in CEM/VLB100 cells and was only moderately increased in HL-60/AR cells. In contrast, when cells were transferred to verapamil-containing medium, vincristine activity was greatly increased against both CEM/VLB100 and HL-60/AR cells. Verapamil enhanced accumulation and inhibited release of [3H]vincristine by CEM/VLB100 and HL-60/AR cells, indicating that the sensitization was due to an increase in cell-associated vincristine after transfer of cells to vincristine-free medium. Slot blot analysis of cellular RNA with the pMDR1 probe revealed high levels of expression of the mdr1 gene in CEM/VLB100 cells but no detectable expression in HL-60/AR cells. Consistent with this finding, polypeptides (Mr 170,000 to 180,000) that were recognized by a monoclonal antibody (C219) against P-glycoprotein were greatly overexpressed in CEM/VLB100 cells, but were expressed at low levels, if at all, in HL-60/AR cells. These results demonstrate the importance of duration of exposure to verapamil in reversing multidrug resistance, not only in cells that overexpress P-glycoprotein but also in cells, such as HL-60/AR, that express little, if any, P-glycoprotein.