The extent of the F plasmid oriT nicking region was determined from the properties of successive substitution mutations in the region from base pair 121 to base pair 174 and from KMnO4 probing of DNA structural distortions induced in vivo by tra gene products. Nicking and transfer assays indicated that the left margin of oriT lies predominantly at the nick site, and that the nicking domain primarily lies within 17 bp to the right of the nick. Some mutants that were proficient for nicking showed reduced frequencies of termination, indicating that oriT nicking does not guarantee efficient termination. DNA in the vicinity of the nick (G137, T138, G140, and T141 on the nicked strand) showed elevated sensitivity to KMnO4 when tra gene products were present in the donor. Bases C145, C146, C147, C149, and G150 on the unnicked strand also became more sensitive to oxidation under tra+ conditions. The bases preferentially oxidized by KMnO4 lie within the nicking domain, as defined by the substitution mutants, and they include dinucleotides that can produce kinks in the DNA. Base pairs in the nicking region are calculated to be more thermodynamically stable than base pairs in the flanking regions.