Gamma rays have been used to induce Chinese hamster ovary cell mutants in which the entire locus for dihydrofolate reductase (DHFR) has been eliminated. These mutants were isolated in two steps from a methotrexate-resistant clone (Flintoff, Davidson, and Siminovitch (1976). Somat. Cell Genet. 2, 245-262). The resistant cells contain amplified copies of a mutant dhfr gene that codes for a drug-resistant form of the enzyme. In the first step, methotrexate-sensitive mutants of the amplified line were selected. These mutants exhibit a reduced level of DHFR activity and contain a reduced number of dhfr genes. The remaining DHFR activity is methotrexate-sensitive. These mutants appear to be hemizygotes that have lost all copies of the amplified altered dhfr genes and retain one wild-type allele. In a second mutagenic step, mutants completely deficient in DHFR activity were isolated. Three of four of these mutants were the result of double deletions: they lack all traces of dhfr DNA sequences. The fourth mutant contains a deletion that extends through the 5' half of the dhfr gene. The hemizygotes for dhfr should be useful for the study of mutation at an autosomal mammalian locus without the complications of diploidy.