Previous studies have demonstrated that herpes simplex viruses (HSV) type 1 (HSV-1) and type 2 (HSV-2) can be maintained in a repressed form in human embryo lung cells. Reducing the incubation temperature or superinfecting with a heterologous herpesvirus, human cytomegalovirus (HCMV), results in activation of virus replication. We now report that superinfection with a partially homologous herpesvirus, HSV-2, also resulted in activation of HSV-1. To minimize excessive synthesis of infectious HSV-2 while allowing virus gene expression, repressed HSV-l-infected cultures were superinfected with HSV-2 temperature-sensitive (ts) mutants (tsF3, tsB5, or tsH9). The predominant virus replicated after HSV-2 ts mutant superinfection at a nonpermissive temperature was identified as activated parental-like HSV-1 by (i) plaquing efficiency at permissive (34 degrees) and nonpermissive (40.5 degrees) temperatures, (ii) sensitivity to inhibition by the HSV-l-specific antiviral agent (E)-5-(2-bromovinyl)-2'-deoxyuridine, and (iii) restriction endonuclease cleavage analysis. In addition, the fact that superinfection with HSV-2 tsB5 or tsH9, which are unable to synthesize virus DNA and express only early virus genes at nonpermissive temperature, resulted in synthesis of virus demonstrated that HSV-2 DNA synthesis is not required for activation. This system has provided the basis for further studies concerning the regulation of HSV gene expression in human cells.