We have previously described a series of methotrexate (MTX)‐selected CCRF‐CEM sublines (CEM/MTX R1–3) displaying increased resistance to drugs associated with the multidrug resistance phenotype and have provided evidence that MDR1 P‐glycoprotein contributes to multifactorial MTX resistance in these cells. We have also suggested that P‐glycoprotein‐mediated MTX transport arises in these cells due to a deficiency in the normal MTX transport route, the reduced folate carrier (RFC). We have now determined the nucleotide sequence of the RFC gene in CEM/MTX R1–3 cells and confirm that the carrier is defective in these cells as a result of a premature stop mutation at codon 99, which severely truncates the encoded protein. CEM/MTX R3 cells were removed from MTX, and a series of sublines with increasing MDR1 expression were derived, following selection with vincristine. These cells show increasing cross‐resistance to vincristine as well as other drugs associated with the multidrug resistance phenotype. More importantly, the increased P‐glycoprotein expression correlates with increased resistance to MTX, supporting the hypothesis that in cells with a defective carrier protein, MTX can become a substrate for P‐glycoprotein. Our data have implications for the P‐glycoprotein‐mediated transport of other hydrophilic drugs in situations where the relevant carrier protein has been functionally inhibited. Int. J. Cancer 78:176–181, 1998.© 1998 Wiley‐Liss, Inc.