Multidrug resistance-associated protein 1 and P-glycoprotein are major ATP-binding cassette transporters that function as efflux pumps and confer resistance to a variety of structurally unrelated anticancer agents. To evaluate the comparative importance of these transporters with respect to anticancer agents, we established and characterized SV40-immortalized [mrp1(-/-)] (KO), [mdr1a/1b(-/-)] (DKO), and combined [mrp1 (-/-), mdr1a/1b(-/-)] (TKO) deficient fibroblast lines derived from primary embryonic fibroblasts of knockout mice. Western blot analyses demonstrated that KO and DKO fibroblasts exhibited similar levels of P-glycoprotein and mrp1, respectively, to that of wild-type (WT) fibroblasts. In addition, semiquantitative reverse transcription-PCR measurements of other multidrug resistance-associated protein (mrp) family members demonstrated that TKO fibroblasts displayed expression profiles of mrps 2-7 comparable to that of WT fibroblasts. These results indicate that loss of mrp1, P-glycoprotein, or both transporters does not cause overt compensatory changes in the expression of the other determined transporters. Using cell viability and calcein accumulation assays, we demonstrated that KO and DKO fibroblasts exhibited a low to moderate increase in sensitivity to vincristine and etoposide and in calcein accumulation compared to WT fibroblasts, whereas TKO fibroblasts displayed a markedly enhanced sensitivity to these agents and further elevated calcein accumulation. Furthermore, verapamil, an inhibitor of both mrp1 and P-glycoprotein, significantly sensitized WT fibroblasts to both vincristine and etoposide while having no effect on the sensitivity of TKO cells to these agents. Collectively, these findings indicate that mrp1 and P-glycoprotein are major determinants of drug sensitivity in immortalized mouse embryonic fibroblasts. They also suggest the existence of a compensatory mechanism by which the loss of one transporter can be functionally offset by the other in the transport of common drug substrates.