Anthramycin and mitomycin C (MC) are two DNA reactive drugs, which bind covalently to GC pairs producing different effects on DNA: anthramycin stiffening and MC distorsion. This paper describes experiments in which we have used anthramycin as a probe to sense quantitatively the effects on DNA of MC binding. Saturation binding experiments show that both anthramycin and MC partially inhibit the binding of the other drug to DNA (maximum inhibition by MC and anthramycin, 22.4% and 19.7%, respectively) but by a mechanism other than direct site exclusion. This suggests that MC binds in the major groove of DNA, since anthramycin is known to bind in the minor groove. An abrupt reduction in the binding of anthramycin to DNA-MC complexes occurs between MC binding ratios of 0.030 and 0.035, which parallels and probably results from sudden intensification of a MC-induced DNA conformational change occurring between these binding ratios. Dialysis measurements indicate that anthramycin is very possibly binding at sites distant from MC sites and suggest a clustering of closely bound MC chromophores resulting from possible cooperative binding. S1 nuclease digest experiments demonstrate an initial enhancement of nuclease activity in DNA-MC complexes, the magnitude of which correlates well with the reduction of anthramycin binding, relative to the degree of MC binding. The enhanced nuclease activity in these complexes indicates regions of exposed DNA or helix base distortion which is related to or is the result of conformational change.