Endonuclease V of bacteriophage T4 has been purified to physical homogeneity from T4D-infected Escherichia coli 1100. The enzyme, whose molecular weight is 16,000, possessed two distinct catalytic activities, a pyrimidine dimer-DNA glycosylase and an apurinic/apyrimidinic endonuclease. They acted on UV-irradiated poly(dA) . poly(dT) in a sequential manner; the glycosylase cleaved the N-glycosyl bond between the 5'-pyrimidine of a dimer and the corresponding sugar and then the endonuclease hydrolyzed a phosphodiester bond on the 3'-side of the apyrimidinic site. The 5'-termini thus generated were phosphorylated by T4 polynucleotide kinase only after they had been subjected to direct photoreversal and then treated with alkaline phosphatase. By using two phage mutants, uvs-5 and uvs-13, it was shown that occurrence of an amber mutation in the denV gene caused a simultaneous loss of the two activities. Suppression of the mutation of uvs-5 rendered both activities partially active. When the mutation of uvs-13 was suppressed, a mutant form of enzyme that possessed only a glycosylase activity was produced. This suggests that there are two distinct domains in a single enzyme, each of which corresponds to one of the activities.