A highly resistant strain of the L5178Y mouse leukemle, able to survive continuous treatment with 500 µg/ml methotrexate (MTX), was obtained in vitro by continued treatment of cultures with increasing amounts of MTX. Measurements of dihydrofolate reductase in resistant cultures treated with MTX showed that enzyme levels were about 100 times higher than those in untreated cultures of the sensitive strain. During growth without MTX, both enzyme levels and resistance declined. At about 100 days after termination of treatment, enzyme levels were similar to those of sensitive cells. The growth rates of sensitive and resistant cultures did not differ and the enzyme level declined even in cultures cloned at the start, showing that this loss of resistance was not the result of cell selection processes. Scoring of the survival of clones in soft agar with different concentrations of MTX indicated that, after a period of growth without MTX, the cultures contained a mixture of cells of differing degrees of resistance. When treated with MTX, less resistant clones had partly or completely lost their ability to synthesize increased amounts of dihydrofolate reductase. This result appears to exclude the possibility that the enzyme level declined because of some physiological adaptive process in the cells. It suggests that the loss of resistance is due to the instability of the mutation controlling dihydrofolate reductase production. The rate of reversion from full resistance was 5 × 10-2.