Apurinic sites are position-specific poisons of topoisomerase II and stimulate DNA scission approximately 10-18-fold when they are located within the 4-base overhang generated by enzyme-mediated cleavage (Kingma, P. S., and Osheroff, N. (1997) J. Biol. Chem. 272, 1148-1155). To determine whether other major forms of spontaneous DNA damage also act as topoisomerase II poisons, the effects of position-specific apyrimidinic sites and deaminated cytosines (i.e. uracil:guanine mismatches) on the type II enzyme were determined. Both of these lesions stimulated topoisomerase II-mediated DNA scission with the same positional specificity as apurinic sites but were less efficacious. Moreover, apurinic sites dominated the effects of apyrimidinic sites in substrates that contained multiple lesions. The differential ability of spontaneous lesions to enhance DNA cleavage did not correlate with either a decreased stability of the double helix or the size of the gap formed by base loss. Rather, it appears to be due (at least in part) to increased rates of religation for substrates containing apyrimidinic sites or deaminated cytosines. These results suggest that several forms of spontaneous DNA damage are capable of acting as endogenous poisons of topoisomerase II.