Excision tracts generated in cell extracts by using circular substrates containing a mismatch and a single GATC site. The covalently closed circular heteroduplex contained a G-T mismatch. Since hemimethylation imposes an asymmetry on the helix, heteroduplexes are designated according to the orientation of the unmodified strand. During the reactions in the cell extracts, DNA resynthesis was blocked by dideoxynucleoside triphosphates. Electron microscopic visualization of DNA products produced under these conditions revealed the presence of single-strand gaps that spanned the shorter path between the mismatch and the GATC site in the circular substrate.