Ultraviolet irradiation of DNA in vitro results in the production of a wide variety of pyrimidine base alterations, including cytosine hydrates. Enzymes that initiate the repair of monomeric pyrimidine damage have been identified in both bacterial and mammalian systems; however, the in vivo formation and repair of cytosine photohydrates has not been demonstrated in cellular DNA. Using Escherichia coli endonuclease III as a damage-specific probe, we have shown that ring-saturated pyrimidines are formed in cultured human cells by irradiation with broad-spectrum UV light. In addition, these types of base damage are removed from the DNA of human lymphoblasts within 5 h following the irradiation. Analysis of the action spectrum for the formation of cytosine hydrates in DNA reveals that these photoproducts are formed most efficiently by irradiation in the range of 255-265 nm light, coinciding with the wavelengths that are maximally absorbed by the DNA bases.