Spontaneous mutants of Saccharomyces cerevisiae able to incorporate deoxythymidine-5'-monophosphate (dTMP) into deoxyribonucleic acid (DNA) have been selected based on their ability to grow in the presence of aminopterin and sulfanilamide if dTMP is present. Essentially all mutants (called tup) selected in this way required dTMP for growth in the presence of the two drugs, but none required dTMP in the absence of the drugs. Neither thymine nor thymidine would satisfy this requirement. Equimolar amounts of (32)P- and (3)H-base-labeled dTMP were incorporated by the mutants into alkali-stable, deoxyribonuclease-sensitive material. In the presence of aminopterin and sulfanilamide, this incorporation was sufficient to account for a substantial proportion of the thymine residues in the cellular DNA, whereas in the absence of the drugs only about 40% as much of the thymine residues originated from the medium. Of 29 mutants examined, all were recessive and 17 showed 2:2 segregation in crosses with a wild-type strain. The lesions in these mutants fell into four complementation groups: one (tup1) occurs on chromosome III; another (tup3) is on chromosome II; and a third (tup4) was centromere linked. Strains of the genotype alpha tup1 mated with lower than normal efficiency with a strains, but with higher than normal efficiency with alpha strains. Strains of genotype a/alpha tup1/tup1 failed to sporulate, whereas homozygous diploids for tup2, tup3, or tup4 sporulated normally, as did a/alpha tup1/+ strains.