Rat embryo cells were treated with 3H-labeled 5-bromodeoxyuridine (BrdU) in order to determine the nature of substitution and distribution of the analog in DNA. At optimal oncornavirus-inducing doses of BrdU (10-4 M), density gradient centrifugation and DNA·DNA reassociation experiments revealed extensive (>90%) and uniform base substitution in repetitive, intermediate, and unique DNA sequences. Suboptimal doses (10-7 M) of [3H]BrdU resulted in less than 5% thymine replacement and nonrandom labeling of DNA. Treatment of cells with 10-7 M [3H]dT resulted in relatively uniform labeling throughout all DNA sequences. At low concentrations, BrdU was incorporated predominantly within the repetitive and intermediate DNA of rat embryo cells. Moreover, the single-copy DNA sequences were lightly substituted and reassociated extensively only in the presence of excess unlabeled DNA. Such specificity of base substitution may be related to the unusually selective nature of BrdU effects on animal cells.