Certain chemical compounds increase mutation frequency of Escherichia coli B/r significantly when used in conjunction with nonlethal ultraviolet (UV) dosages. Studies were done to elucidate the mechanism of this enhancing mutational effect. Dark survival curves showed that 500 ,ug of caffeine per ml in the postirradiation medium markedly decreased survival to 60 ergs/mm2 of UV in strain B/r. Caffeine did not markedly decrease survival to UV in strain B/r WP-2 hcr-. At least 90% of the mutations induced to streptomycin resistance by UV and 85 % of those induced by UV with caffeine could be photoreversed. Experiments with thymine analogues suggested that thymine dimerization at the streptomycin locus was the primary premutational photoproduct induced by sublethal UV dosages. Caffeine did not interfere with the photoreversal of induced mutants, indicating that it probably does not bind to the photoreactivating enzyme or to a UV-induced lesion in the DNA. Addition of DNA or irradiated DNA with 500 ,g of caffeine per ml resulted in no loss of the caffeine activity. The excision of UV-induced thymine-containing dimers from E. coli B/r Twas investigated in the presence and absence of caffeine. Our results indicated that caffeine prevents excision of thymine dimers, presumably by binding to the excising enzyme. This binding results in an impairment of repair, which produces the increase in mutant numbers. Caffeine, theophylline, and theobromine, as well as acridine dyes, increase the frequency of mutations induced by lethal and sublethal ultraviolet (UV) light when they are added to the postirradiation medium of Escherichia coli strain B/r (3, 14, 23, 29, 30). Bacterial killing is increased when bacteria irradiated with lethal and sublethal dosages are plated on a medium containing acriflavine (1, 7, 30) or caffeine (7, 17, 29). These compounds decrease the survival of UV-irradiated phage by inhibiting host cell reactivation (5). The increase in both killing and mutation can be attributed to inhibition of a "dark repair" process that repairs UV photoproducts in the irradiated bacteria. Irradiation of bacteria with high dosages of UV light produces a variety of pyrimidine dimers in the deoxyribonucleic acid of these organisms (DNA) (21). Pyrimidine dimers can be repaired either by photoreactivation, in which the photoreactivating enzyme splits dimers in situ (35), or by excision of pyrimidine dimers from the DNA in the dark (2, 20, 25). Numerous UVsensitive mutants of E. coli have been isolated 198 Vol. 96, No. I