Stable mutants of Chinese hamster V79 cells deficient in thymidylate synthetase (TS; E.C. 18.104.22.168) have been selected from cultures grown in medium supplemented with folinic acid, aminopterin, and thymidine (FAT). After chemical mutagenesis, the frequency of colonies resistant to the “FAT” medium increased more than 100-fold over the spontaneous frequency. The optimal expression time of the mutant phenotype was 5–7 days after mutagen treatment. The recovery of FAT-resistant colonies in the selective medium was not affected by the presence of wild-type cells at a density below 9,000 cells per cm 2 . All 21 mutants tested exhibited thymidine auxotrophy; neither folinic acid nor deoxyuridine could support mutant cell growth. There was no detectable TS activity in all 11 mutants so far examined and only about 50% of wild-type activity in three prototrophic revertants, as measured by whole-cell and cell-free enzyme assays. The apparent Michaelis-Menten constant (K m ) for deoxyuridine-5′-monophosphate and inhibition constant (K i ) for 5-fluorodeoxyuridine-5′-monophosphate, measured by whole-cell enzyme assay, appear to be similar for the wild-type and revertant cell lines. Using 5-fluoro-[6 3 H]-2′-deoxyuridine 5′-monophosphate as active site titrant, the relative amounts of TS in crude cell extract from the parental, revertant, and mutant cells were shown to exist in a 1:0.5:0 ratio. Furthermore, the enzymes from two revertants were more heat labile than that of V79 cells. These properties, taken together, suggest that the FAT-resistant, thymidine auxotrophic phenotype may be the result of a structural gene mutation at the TS locus. The availability of such a mutant facilitates studies on thymidylate stress in relation to DNA metabolism, cell growth, and mutagenesis.