The cytotoxic effect of the combination of N-methylformamide (NMF) with 5-fluorouracil (5-FU) on cell survival of the human colon cancer line HT29 was assessed. The differentiating activity of NMF was evidenced by morphological maturation and conversion of cell culture characteristics to those consistent with a more benign phenotype. In combination experiments, the noncytotoxic concentration of 1% NMF was chosen and doses of 5-FU ranging from 5 to 25 micrograms/ml were employed. Two main schedules were tested either on exponentially or stationarily growing cells: (a) 1% NMF for 72 h followed by 12-h exposure to 5-FU; (b) 5-FU for 12 h followed by 72-h exposure to 1% NMF. The results obtained demonstrated that the 5-FU----NMF sequence determined a powerful reduction in the surviving fraction of HT29 cells, while the reverse sequence did not increase the killing effect of 5-FU given alone. Immunocytochemical and scanning electron microscopy studies seemed to confirm that the association in which the differentiating agent followed the 5-FU treatment strongly impaired cellular integrity and function and that cytoskeletal elements, particularly microfilaments, and surface structures could play an essential role in the mechanisms of cytotoxicity. Furthermore, the results of this work indicate that drug sequence is a critical factor for the optimal combination of 5-FU and NMF.