BHK cells infected with the temperature-sensitive mutant ts13 of herpes simplex virus type 2 at a nonpermissive temperature lack the alkaline nuclease activity, which is induced by the mutant at a permissive temperature and by wild-type virus at either temperature. For ts13, enzyme activity could be induced by a temperature shift to permissive conditions, but not in the presence of cycloheximide. After a shift from permissive to nonpermissive conditions in the presence of cycloheximide, the activity was stable in wild-type, but not in mutant-infected, cells. After extensive purification, the wild-type nuclease was fourfold more heat stable in the presence of substrate than was the mutant enzyme. Mixtures of both purified enzymes showed the predicted intermediate stabilities. The results strongly suggest that the enzyme is virus coded and that the mutant possesses a lesion in the structural gene of the enzyme.