The effects on enzymatic DNA repair of aphidicolin and 1-beta-D-arabinofuranosylcytosine (araC), two potent inhibitors of long-patch excision repair, were investigated in cultured human cells exposed to 60Co gamma-radiation. Using alkaline-sucrose velocity sedimentation analysis, both drugs were shown to inhibit markedly the repair of radioproducts in cultures exposed to greater than or equal to 150 Gy, indicating that a significant component of gamma-ray-induced DNA damage is operated on by a long-patch excision pathway. Moreover, while the extent of repair inhibited by aphidicolin was comparable to that suppressed by araC, combined exposure of irradiated cultures to the two drugs elicited a synergistic response. Specifically, in all three normal fibroblast strains examined, the yield of aphidicolin- or araC-detectable sites (lesions whose repair could be blocked by each drug alone) observed during the first 2 h after irradiation with 150 Gy ranged from 0.8 to 1.2 per 10(8) daltons genomic DNA, whereas the incidence of sites detected by combined exposure to the inhibitors was increased 4-fold (i.e. 3.8 per 10(8) daltons). This difference in site yield leads us to propose that simultaneous administration of aphidicolin and araC serves to block, in addition to long-patch repair, a second mode of excision repair which is refractory to each drug alone.