A series of CCRF-CEM sublines selected for extreme resistance to methotrexate has been shown previously to exhibit cross resistance to a number of agents belonging to the multidrug resistance phenotype. The mechanism(s) underlying resistance to vincristine, vinblastine and actinomycin D in the most resistant subline (CEM/MTX R3) has now been investigated. Efflux of [3H]vincristine was more rapid in CEM/MTX R3 than in either CCRF-CEM cells or a methotrexate-resistant subline not refractory to Vinca alkaloids. In addition, verapamil completely reversed resistance to vincristine, vinblastine and actinomycin D in the CEM/MTX R3 cells. While these results are suggestive of P-glycoprotein-mediated multidrug resistance, Northern analysis revealed no detectable expression of the mdr 1/gene in CEM/MTX R3 cells. Likewise, karyotypic analysis of the resistant subline, while revealing certain clonal abnormalities, provided no evidence of alteration in the mdr 1/gene locus on chromosome 7. The data suggest therefore the operation, in these cells, of a novel mechanism of resistance.