Chemical probes sensitive to alterations in DNA conformation, especially Z-DNA, have been identified. These permit cleavage of DNA at sites of unusual structure, the results of which can be displayed on a sequencing gel. Using supercoiled plasmids containing inserts of d(C-G)16 and d(C-A)31 X d(T-G)31, it was found that hydroxylamine and osmium tetraoxide react preferentially with cytosines and thymines, respectively, near B-DNA-Z-DNA junctions; diethylpyrocarbonate reacts more strongly with purines within Z-DNA regions; and dimethylsulfate and diethylsulfate react more strongly with guanines in Z-DNA that are out of phase with the usual pattern of purine-pyrimidine alternation. Our results show that B-Z boundaries are mobile and that with increasing torsional strain, the Z-DNA regions can expand to include nonalternating nucleotide sequences.