Ultraviolet light induces alkaline labile lesions in DNA. These lesions occur at the bipyrimidine sites T-C, C-C, and T-T, and do not result from the formation of pyrimidine cyclobutane dimers. To examine the chemical nature of the alkaline labile lesions, pyrimidine dinucleotides (2'-deoxythymidylyl-(3' leads to 5')-2'-deoxycytidine, 2'-deoxythymidylyl-(3' leads to 5')-2'-deoxythymidine, 2'-deoxycytidylyl-(3' leads to 5')-2-deoxycytidine, and 2'-deoxycytidylyl-(3' leads to 5')-2'-deoxythymidine) were used as a model system. Ultraviolet light irradiation of all four dinucleotides resulted in the formation of substantial quantities (relative to cyclobutane pyrimidine dimers) of products that were red-shifted in UV absorbance and fluorescent. These products are identified as precursors to the 6-4'-[pyrimidin-2'-one]-pyrimidine class of products that have been previously shown to occur in UV-irradiated DNA. The fluorescent products were found to be alkaline labile; the products contained a 3'-phosphate end group after alkali treatment. Two of the fluorescent products have been found in enzymatic digests of high dose UV-irradiated salmon sperm DNA, the T-C and T-T UV-induced products. The relative rates of formation of these products in 32P-radiolabeled dinucleotides were measured. We conclude that the alkaline labile lesions observed in DNA at the bipyrimidine sites are the same as those that yield the 6-4 products upon acid hydrolysis of DNA. The mechanisms for the formation and the consequences of DNA structure for these lesions as well as the possible biological significance of this class of UV damage are discussed.