ABSTRACT Thymine deprivation can be achieved in bacteriophage T4 either by the use of the thymidylate synthetase inhibitor FUdR, or by an appropriate combination of genetic blocks; both methods produce marked mutagenesis. Extensive tests of the specificity of thymineless mutagenesis reveal that only A:T base pairs are affected, and that transitions and possibly transversions are produced. This system therefore constitutes the fist example of an A: T-specific mutagen. Thymineless mutagenesis in bacteriophage T4 exhibits a marked dependence upon the functional state of the DNA polymerase gene, but is largely independent of the px-γ misrepair system.