The influence of caffeine on the activity of DNA polymerase I from E. coli was investigated. Caffeine had no effect on the polymerizing activity but did inhibit both 5' leads to 3' and 3' leads to 5' nuclease activities. The highest inhibition was observed with d(A--T)n as substrate: at a concentration of caffeine of 10 mM, inhibition was about 50%. In studies in vivo with 3 isogenic strains of E. coli, carrying different mutations in the DNA polymerase I gene, the effect of caffeine on survival after ultraviolet irradiation was most marked for the wild-type, pol+, followed by those mutants defective in 3' leads to 5', polA1, and 5' leads to 3' nuclease activities, polA107. Caffeine had little influence on survival of the resA1 mutant which lacks both 5' leads to 3' and 3' leads to 5' nuclease activities. These results support the idea that the influence of caffeine on dark repair may be explained in part by its effect on the nuclease activities of DNA polymerase I.