Cultures of human colon carcinoma, HCT-8, were treated with millimolar concentrations of thymidine by different schedules designed to cytokinetically and biochemically modulate methotrexate (MTX) and 5-fluorouracil (FUra) toxicity. Thymidine (dThd)-synchronized HCT-8 cells monitored by flow cytofluorometry showed increased sensitivity to MTX and synergistic cytotoxicity to the combination MTX-FUra. FUra toxicity in synchronized cells showed no significant phase specificity overall, but a pattern of relative G2/M resistance was correlated with decreased intracellular FUra accumulation, incorporation into RNA, and formation of FdUMP. In asynchronous cultures dThd reduced MTX toxicity when given within the first 12 h of a 24-h MTX exposure, and also appeared to reduce the MTX-induced synergistic enhancement of FUra toxicity. When dThd was administered with FUra alone in asynchronous cultures, progressive, and synergistic enhancement of FUra toxicity was observed only after 6 h dThd pretreatment. Unlike MTX-FUra synergy, this schedule-dependent synergism between dThd and FUra did not correlate with intracellular FUra accumulation or specific incorporation into total cellular RNA. These results suggest that less well studied mechanisms of dThd modulation, other than enhanced deoxynucleotide formation or total RNA incorporation, may biochemically enhance FUra toxicity in HCT-8 cells.