A spontaneously arising clone, stably resistant to the toxic effects of the thymidine analog, 5-hydroxymethyl-2'-deoxyuridine (hmdU), was isolated from unmutagenized V79.5 Chinese hamster fibroblast cells by a single-step selection procedure. The hmdUr cells were selected in the continuous presence of 30 microM hmdU, a concentration which reduces the plating efficiency of wild-type cells to less than 1% after a 24-h exposure. A line of human HeLa cells were found to be intrinsically resistant to concentrations of hmdU as high as 100 microM. All of the hmdUr cells were found to grow normally in HAT medium, which requires the expression of thymidine kinase activity; be sensitive to the toxic effects of high concentrations of 5-bromo-2'-deoxyuridine, another thymidine analog; have unaltered hmdU nucleotide metabolism, as measured by HPLC analysis of acid-soluble cell extracts; and have decreased levels of hmdU incorporation into DNA. Although high concentrations of 5-hydroxymethyluracil (hmUra) were found to be nontoxic for both wild-type and hmdUr cells, the resistance phenotype could be suppressed by exposing the cells to hmdU and high concentrations of hmUra simultaneously.