The relationship between purified transcription factor p50 binding and ultraviolet light-induced DNA damage formation in the NF-kappa B promoter element was investigated. The effect of bound transcription factor on cyclobutane dimer formation was quantified using Maxam-Gilbert analysis of irradiated substrate digested with T4 phage endonuclease V. Two methods were employed for cleaving (6-4) photoproducts. Sites of (6-4) photoproducts cleaved by piperidine showed a general suppression in the presence of bound p50 protein similar to that observed for cyclobutane dimers. In contrast to piperidine, digestion with ultraviolet damage endonuclease (UVDE) from Saccharomyces pombe subsequent to cyclobutane dimer reversal by photolyase displayed a broader spectrum of damaged sites. Whereas some of these sites were suppressed by bound p50 protein, some remained unaffected and one site showed increased (6-4) photoproduct induction. These data illustrate the advantage of UVDE over piperidine for studying (6-4) photoproducts at the sequence level and suggest that this approach may be useful for footprinting transcription factor binding in other promoters.