The anatomy of DNA synthesized by five HSV-1 mutants previously shown to accumulate predominantly empty capsids at the nonpermissive temperature (NPT) was analyzed with Bg/II restriction digestion. At the NPT, all five generated DNA lacking termini, indicating that in the absence of packaging, viral DNA is not processed to unit length. One mutant, F18, was able to process DNA made at the NPT to unit length molecules during a 6-hr period after shift to the permissive temperature. The appearance of unit length molecules correlated with the appearance of staphylococcal nuclease-resistant F18 DNA.