The processing of newly replicated concatameric T5 DNA into both single stranded DNA changed of unit length and single-stranded fragments of sizes comparable to those found in mature T5 virion DNA occurs in the absence of late T5 protein synthesis. The formation of unit-length, single-stranded DNA chains does not require the early T5 gene D15 nuclease: however, the subsequent formation of the single-stranded fragments does require that the D15 nuclease be functional. A reexamination of the properties of the purified D15 nuclease under a variety of conditions showed that, in addition to functioning as a 5' leads to 3' exonuclease, the enzyme can also introduce endonucleolytic scissions into mature T5 DNA in a reaction that requires duplex T5 DNA and preexisting, single-stranded interruptions.