5-Hydroxymethyl-2'-deoxyuridine (HmdUrd), a cytotoxic analogue of thymidine, has been proposed for use as an anticancer agent. HmdUrd is incorporated into DNA and then removed at a rate of 30-40% per day. The removal of HmdUrd from DNA has been attributed to the action of 5-hydroxymethyluracil-DNA glycosylase (HmUra-DNA glycosylase). We demonstrated the release of [3H]HmUra into the growth medium of V79 Chinese hamster cells that had incorporated [3H]HmdUrd into their DNA. The amount of [3H]HmUra recovered from the growth medium was equal to the amount of [3H]HmdUrd lost from DNA. These experiments confirmed that the initial step of repair of HmUra in DNA was mediated by DNA glycosylase activity. A combination of HmUra and HmdUrd resulted in increased uptake of HmdUrd by cells and increased cytotoxicity. The increased incorporation of HmdUrd into DNA was not due to inhibition of repair. 3-Aminobenzamide, an inhibitor of poly(ADP-ribose) synthesis, was cytotoxic to cells which incorporated and repaired HmdUrd. The extent of toxicity was directly related to the number of HmUra residues in DNA. HeLa cells, known to be resistant to the toxic effects of HmdUrd, do not incorporate HmdUrd into their DNA. HeLa cells were resistant to the toxic effects of 3-aminobenzamide, confirming that the absence of HmdUrd in their DNA was not due to an accelerated rate of repair. These experiments indicate that the potential therapeutic antineoplastic properties of HmdUrd may be enhanced by using HmUra to increase the incorporation of HmdUrd into DNA and 3-aminobenzamide to interfere with repair of HmUra in DNA.