The methotrexate-resistant AT-3000 line of S-180 cells has at least 150-fold more immunologically cross-reactive folate reductase than sensitive cells. Highly specific immunologic and protein purification procedures were used to show that the increased enzyme levels in this line are due to a corresponding increase in the rate of folate reductase synthesis. This observation indicates that the relative turnover of the enzyme is not significantly different in the two lines. Folate reductase was purified to homogeneity from both the sensitive and the methotrexate-resistant cells. Comparison of various physical, kinetic, and immunochemical properties of the enzymes revealed no differences. These observations suggest that the AT-3000 line contains one or more regulatory variations leading to the over-production of folate reductase protein that is similar, if not identical, to that produced by sensitive cells. In resistant cells, specific immunoprecipitation experiments demonstrated that folate reductase comprises as much as 7 to 8% of the continuously labeled soluble protein and 6 to 7% of the soluble protein synthesis. Growth of these lines in the absence of methotrexate resulted in a slow decrease in the level of folate reductase to less than 1%. This decrease corresponded to a similar decrease in the relative rate of enzyme synthesis. Variations in the level of folate reductase with cell growth are also due to changes in the relative rate of enzyme synthesis. In the AT-3000 line, pulse decay experiments showed that the half-life of folate reductase was long (50 hours) relative to cell doubling time (24 hours), and also that methotrexate had little or no effect on the turnover of the enzyme. Comparison of the incorporation of radioactive leucine into folate reductase in continuous and pulse labeling experiments gave independent confirmation of these results. Therefore, the relative rate of folate reductase synthesis was the major parameter determining the amount of folate reductase under all examined conditions that resulted in altered levels of the enzyme in resistant cells.