BACKGROUND The anti-P-glycoprotein monoclonal antibody MRK-16 mediates the reversal of multidrug resistance. Recombinant human interferon alfa (rHuIFN alpha) enhances the cytotoxic activity of diverse chemotherapeutics and may modulate multidrug resistance. OBJECTIVE Our purpose was to determine the outcome of combination treatment with MRK-16, rHuIFN alpha-2a, and cytotoxic agents on tumor cells that express P-glycoprotein (Pgp). METHODS Three Pgp-expressing, multidrug-resistant human tumor cell lines were used: the MDR1 retrovirus-infected HT-29 colon adenocarcinoma (HT-29mdr1), the doxorubicin (Adriamycin)-resistant MCF-7 (AdrR MCF-7) breast carcinoma, and the de novo Pgp-acquired, HCT-15 colon carcinoma. The parental cell lines HT-29par and MCF-7 were used as controls. The in vitro effects of MRK-16 and rHuIFN alpha-2a were studied on: (a) chemosensitivity of parental and multidrug-resistant cell lines to vincristine, doxorubicin, or paclitaxel (Taxol); (b) intracellular drug concentrations; and (c) Pgp expression. The efficacy of vincristine alone or in combination with MRK-16 and/or rHuIFN alpha-2a was assessed against HT-29mdr1 cells in female, athymic NCr-nu/nu mice. RESULTS For vincristine, the IC50 (i.e., the concentration that causes 50% inhibition of cell growth) was 7.0 ng/mL in HT-29mdr1 cells. Pretreatment of HT-29mdr1 cells with MRK-16 partially restored vincristine sensitivity (IC50 = 4.8 ng/mL), which was enhanced by noncytotoxic concentrations of rHuIFN alpha-2a (IC50 = 2.9 ng/mL) via a mechanism independent of Pgp modulation or [3H]vincristine efflux. rHuIFN alpha-2a potentiated MRK-16 reversal of multidrug resistance with both doxorubicin and paclitaxel on HT-29mdr1 cells and with vincristine on AdrR MCF-7 and HCT-15 tumor cells. Treatment of mice with 1 mg/kg vincristine weekly for 3 weeks, beginning 10 days after tumor injection, significantly increased the median survival times of the HT-29par tumor-bearing mice (60 days versus 35 days; P < .0001) but was only marginally therapeutic for HT-29mdr1 tumor-bearing mice (52 days versus 46 days). Pretreatment with MRK-16 (500 micrograms) and rHuIFN alpha-2a (5 x 10(4) U), alone or in combination, 24 hours before vincristine therapy did not affect the survival of HT-29par tumor-bearing mice. In contrast, the survival of mice bearing HT-29mdr1 tumors was significantly increased following treatment with MRK-16 before vincristine (80 days; P < .0001). Administration of a nontherapeutic dose of rHuIFN alpha-2a (5 x 10(4) U) with MRK-16 before vincristine treatment further increased the median survival times of HT-29mdr1 tumor-bearing mice (116 days; P < .0001). CONCLUSIONS MRK-16 used in combination with rHuIFN alpha-2a was significantly more effective than MRK-16 in overcoming multidrug resistance.