We have used pulsed-field gel electrophoresis to examine 5-fluorouracil (5FU)-induced DNA double-strand breaks (DSBs), both with and without modulation by IFN-alpha2a (IFNalpha), in HT29 human colon adenocarcinoma cells. Although 24-h treatment with either 10 microM 5FU or 500 units/ml IFNalpha did not result in significant DNA fragmentation, the combination of 5FU + IFNalpha resulted in a significant increase in DNA DSBs versus either drug alone (P < 0.05). The pattern of fragmentation induced by treatment with 5FU + IFNalpha was compared to that induced by gamma-radiation, which generates lesions at random sites, digestion with NotI restriction endonuclease, which cleaves at the specific sequence 5' ellipsis GCGGCCGCellipsis 3', and HhaI restriction endonuclease, which cleaves at the specific sequence 5'ellipsis GCGCellipsis 3'. 5FU + IFNalpha resulted in a specific pattern characterized by the accumulation of fragments of <3 Mb in the absence of fragments of >3 Mb, which differed from that of gamma-radiation and restriction endonuclease digestion. Because neither morphological nor DNA fragmentation characteristic of apoptosis was observed after 5FU + IFNalpha treatment, the nonrandom pattern of DSBs that was observed did not appear to be the result of the initiation of programmed cell death within these cells.