Bipyrimidine cyclobutane dimers and 6-4'-(pyrimidin-2'-one)-pyrimidine photoproducts are the major adducts formed in DNA following exposure to ultraviolet light. The relationship between the type and frequency of UV-induced DNA damage and the effects of such damage on DNA replication were investigated. UV-irradiated M13 phage DNA was employed in polymerization reactions with the Kenow fragment of Escherichia coli DNA polymerase I. The locations and frequencies of polymerase termination events occurring within a defined sequence of M13 DNA were compared with measurements of the locations and frequencies of UV-induced DNA damage of the same DNA sequence by using UV-specific enzymatic and chemical methods. The results indicate that both cyclobutane dimers and (6-4) photoproducts quantitatively block polymerization by DNA polymerase I.