Plasmids containing (C-G)n inserts have been used to study the inhibition of cleavage by restriction endonucleases due to Z-DNA formation in negatively supercoiled plasmids. The enzyme BssHII, which recognizes G-C-G-C-G-C, is strongly inhibited when the insert forms Z-DNA. The BamHI recognition sequence (G-G-A-T-C-C) was placed in four different positions near the B-Z junction and the inhibition of BamHI cleavage was determined as a function of negative superhelical density. Formation of Z-DNA in the (C-G)n insert inhibited cleavage by BamHI when its recognition sequence was located immediately adjacent to the insert or four base pairs away from it. However, no inhibition was found when the BamHI recognition site was eight base pairs away. These experiments help to define the limits of the structural perturbation associated with the B-Z junction.