In L5178Y mouse lymphoblasts, ionizing radiation-induced mutant frequencies were dramatically higher when the genetic marker analyzed was heterozygous (tk+/tk-) than when hemizygous (tk+/tk0 or hprt+/hprt0). In contrast, base-change mutagens induced similar mutant frequencies at heterozygous and hemizygous loci. These results indicate that the majority of radiation-induced mutants harbor multilocus lesions, and that these mutants are poorly recovered when the target gene is in a hemizygous chromosomal region. Dose-rate dependence of radiation-induced mutant frequency was demonstrated at the heterozygous tk locus but not at the hemizygous hprt locus; in a cell line deficient in the rejoining of DNA double-strand breaks (DSBs), no dose-rate dependence was observed for either locus. The majority of TK-/- mutants, whether spontaneous or induced by X, alpha-particle or UV radiation, or by photosensitization, showed loss of the entire active tk allele. The percentage of TK-/- mutants exhibiting inactivation of galactokinase, encoded by the neighboring gk gene, was high in UV repair-deficient cells exposed to UV radiation and in DNA DSB repair-deficient lines exposed to X radiation. Thus the presence of unrepaired DNA lesions, whether DSBs or pyrimidine dimers, appears to result in an increase in the percentage of mutants harboring multilocus lesions.