Caffeine inhibits excision repair and photoreactivation in E. coli in vivo. We used purified E. coli enzymes and DNase I footprinting to study the mechanism of inhibition in vitro. Photolyase binds to pyrimidine dimers in DNA in a radiation-independent process. Upon irradiation of this enzyme-substrate complex with photoreactivating light, pyrimidine dimers are reverted to their constituent pyrimidine monomers. Using an oligonucleotide containing a thymine dimer at a unique site, we found that caffeine associates with the substrate and inhibits photoreactivation by blocking the binding of photolyase to the dimer. ABC excinuclease catalyses early events of excision repair; recognition of covalently modified DNA and incision of the phosphodiester backbone on both sides of the modification. The UvrA subunit is involved in the damage recognition process, which we studied using an oligonucleotide containing a unique psoralen adduct. UvrA binds to the adduct and protects 33 base pairs surrounding the adduct from DNase I digestion. In the presence of caffeine, the DNaseI footprint of UvrA covers the entire oligonucleotide; thus, caffeine promotes the binding of UvrA to undamaged DNA. UvrA subunits "trapped" by caffeine would be unable to catalyze repair. The intercalators ethidium bromide and chloroquine also promoted UvrA binding to DNA, so it may be caffeine's ability to intercalate into DNA that results in the trapping of UvrA. Thus, as a consequence of its interaction with DNA, caffeine inhibits these repair systems in E. coli by two entirely different mechanisms, by promoting the nonspecific binding of the nucleotide excision repair enzyme and by interfering with specific binding of the photoreactivating enzyme.