Using phosphocellulose followed by ss-DNA cellulose chromatography for UvrC purification from overproducing cells, we found that UvrC elutes at two peaks: 0.4 M (UvrCI) and 0.6 M KCl (UvrCII). Both forms of UvrC have a major peptide band with same molecular weight and identical N-terminal amino acid sequences which are consistent with the initiation codon being at -GTG- site. Both forms of UvrC are active in incising UV-irradiated fX-174 RFI DNA in the presence of UvrA and UvrB; however, the specific activity of UvrCII is one-fourth that of UvrCI. The molecular weight of UvrCII is four times of UvrCI based on size exclusion chromatography and glutaraldehyde crosslinking reactions indicating that UvrCII is a tetramer of UvrCI. Functionally, these two forms of UvrC proteins can be distinguished under reaction conditions in which the protein/nucleotide ratio is above 0.06 by using UV-irradiated, 32P-labeled DNA fragments as substrates; under these conditions UvrCII is inactive in incision, but UvrCI remains active. The activity of UvrCII can be restored by adding unirradiated competitive DNA, and the increased incision corresponds to a decreased UvrCII binding to the substrate DNA. The sites of incision around a UV-induced pyrimidine dimer are the same for UvrCI and UvrCII. Nitrocellulose filter binding and gel retardation assays show that UvrCII binds to both UV-irradiated and unirradiated ds-DNA with the same affinity (Ka = 9 x 108 M-1) in a concentration dependent manner, while UvrCI does not. These two forms of UvrC were also produced by the endogenous uvrC operon. We propose that UvrCII-DNA binding may interfere with Uvr(A)2B-DNA damage complex formation. However, because of its low copy number and low DNA binding affinity, UvrCII may not interfere with Uvr(A)2B-DNA damage complex formation in vivo, but instead through ds-DNA binding UvrCII become concentrated at genomic areas and therefore facilitate nucleotide excision repair.