Dihydrofolate reductase was isolated from hamster cells sensitive to methotrexate and from a methotrexate-resistant subline which has an elevated enzyme level. The enzymes were compared by peptide map analysis, and no differences in primary structure could be detected. The rates of enzyme degradation and synthesis were determined in both cell lines by a novel approach based on the enzyme specific radioactivity (called radioaffinity labeling). Degradation of reductase was minimal in both cell lines, whereas the rates of synthesis were directly proportional to the steady state concentrations of enzyme. Thus the resistant cells synthesized reductase at a rate which was 140 times faster than that in sensitive cells. Therefore the high concentration of dihydrofolate reductase in the methotrexate-resistant cells is probably the result of an alteration of a cellular component which control the synthesis of the enzyme.