1-beta-D-Arabinofuranosylcytosine (ara-C) and hydroxyurea (HU) were investigated as potential DNA repair inhibitors with cis-diammine-1,1-cyclobutane dicarboxylatoplatinum(II) (CBDCA). HU plus ara-C, known inhibitors of DNA excision-repair, had previously been found to produce cytotoxic synergy and delayed removal of DNA interstrand cross-links with cis-diamminedichloroplatinum(II) (DDP). Since CBDCA and DDP share a common active intermediate, it should be possible to reproduce this interaction with CBDCA. However, the stable dicarboxylate chelate ring structure of CBDCA results in kinetics that differ significantly from those of DDP, due to slower hydrolysis to the active species. DNA adducts form more slowly, with interstand cross-links peaking approximately 12-h later and disappearing more gradually than in the case of DDP. It was therefore expected that a longer antimetabolite exposure might be required for repair inhibition with CBDCA. The 12-h exposure to HU plus ara-C previously found effective with DDP produced no cytotoxic synergy with a 2-h CBDCA exposure. Lengthening the antimetabolite treatment to 24 h resulted in approximately 1 log of synergistic toxicity, while a 24-h simultaneous exposure to HU, ara-C, and CBDCA resulted in 2 logs. Cells exposed to all three drugs showed a 2- to 3-fold greater level of interstrand cross-links after 36- to 48-h of incubation following drug removal, compared to CBDCA alone. Taken together, these findings suggest that HU plus ara-C modulates the repair of platinum-DNA adducts and establishes an effective in vitro schedule at clinically achievable concentrations for the use of those antimetabolites with CBDCA.