A simple, continuous spectrophotometric assay for dihydrofolate reductase (DHFR) activity was adapted for determination of drug-resistant enzyme activity expressed from transfected genes in cells containing drug-sensitive enzyme. Methotrexate inhibition characteristics in this assay system were assessed for the murine wild-type (WT) enzyme as well as variant genes encoding amino acid substitutions at codon positions 22 (arg22) or 31 (trp31) expressed in DHFR-deficient Chinese hamster ovary (CHO) cells and in mouse 3T3 cells. Methotrexate concentrations were thus identified which maximized inhibition of the wild-type enzyme while maintaining substantial arg22 or trp31 activity. Mixing experiments were conducted to determine the minimum amount of drug-resistant enzyme distinguishable from a constant amount of wild-type enzyme in the presence of methotrexate. Mixtures of enzymes from a variety of sources (WT, arg22, or trp31 expressed in CHO or 3T3 cells) demonstrated a detection limit of 0.03 to 0.06 nmol/min. Assay of methotrexate-resistant arg22 DHFR appeared to be limited by the low level of activity associated with this enzyme variant, whereas assay of the trp31 variant was limited by enzyme inhibition at lower concentrations of methotrexate. The assay was thus applicable to two quite diverse DHFR variants and may be useful for assaying the expression of other drug-resistant DHFR genes as well after introduction into cells containing drug-sensitive enzyme.