V79 cells incorporated 5-hydroxymethyl-2'-deoxyuridine (HmdUrd) into their DNA linearly over a wide range of concentrations and time. Cells grew normally when 0.03% of thymidine residues were replaced with HmdUrd. At this level of substitution, 5-hydroxymethyluracil (HmUra) was removed from DNA at a rate of 30-40%/24 h. Concentrations of HmdUrd in the growth medium which produced higher levels of substitution reduced survival and caused cells to delay their transit through S phase. However, the treatment of HmdUrd-containing cells with 3-aminobenzamide caused extensive cell death. At levels of HmdUrd substitution compatible with near 90% survival, the addition of 3-aminobenzamide, an inhibitor of poly (adenosine diphosphoribose) synthesis, killed over 90% of the cells. This toxicity was not due to inhibition of the removal of HmUra from DNA. Cells killed by this combination of agents arrested in the G2 phase of the cell cycle. We conclude that the toxicity of HmdUrd resulted primarily from the repair of the HmUra residue in DNA and not from any intrinsic toxicity of the HmUra residue itself. We also conclude that the cytotoxicity of 3-aminobenzamide resulted from interference with the completion of DNA repair following base (HmUra) excision. Since HmUra is also formed in DNA through the action of ionizing radiation, it may be among the components of radiation-induced DNA damage which sensitizes cells to 3-aminobenzamide.