An enzyme activity, named T4 endonuclease V, was purified from T4-infected Escherichia coli. The enzyme induces single-stranded breaks in ultraviolet-irradiated DNA but does not act on native or heat-denatured DNA. The enzyme activity is dependent on the dose of ultraviolet irradiation, and the number of the breaks formed is approximately equal to the number of pyrimidine dimers present in the DNA. Denatured DNA, which has been exposed to ultraviolet light, is also attacked by the enzyme although the extent of the reaction is greater with irradiated native DNA. The enzyme shows optimal activity at pH 7.5 and does not require added divalent ions. When the enzyme-treated, irradiated DNA is subjected to stepwise degradation by spleen phosphodiesterase, dimers are released more rapidly than thymine into the acid-soluble fraction, suggesting that the enzyme induces a break at the 5'-side of a pyrimidine dimer. The enzyme, whose formation is controlled by the v(+) gene of T4, appears to be responsible for the first step of excision repair.