The UvrABC nuclease, the nucleotide excision repair complex from Escherichia coli, is able to incise a variety of types of DNA damage and the repair efficiency of this enzyme complex appears to be influenced by the structure of the damage and the sequence context within which the damage is positioned. In order to better establish these relationships, we have constructed two DNA sequences each containing a site-specifically positioned N-2-aminofluorene (AF) or N-acetyl-2-aminofluorene (AAF) adduct and have determined both the kinetics of UvrABC nuclease incision and the kinetics of UvrABC nuclease-substrate complex formation. It is well established that these two adducts induce very different structures in the DNA and that these structures also depend on the sequence context. We have found that the rate of incision of both AAF- and AF-DNA adducts is significantly faster when they are positioned in the mutation hotspot NarI sequence (5-GGCG*CC-3') than when located in a normal or non-NarI sequence (5'-GATG*ATA-3') and that the rate of incision for AAF-DNA adducts is faster that for AF adducts in both sequences. Most siginificantly, we find that the rate of UvrB and UvrBC-substrate complex formation correlates with the rate of UvrABC nuclease incision.