Replication defective mutants of HSV have been proposed both as vaccine candidates and as vehicles for gene therapy because of their inability to produce infectious progeny. The immunogenicity of these HSV replication mutants, at both qualitative and quantitative levels, will directly determine their effectiveness for either of these applications. We have previously reported (Brehm et al., J. Virol., 71, 3534, 1997) that a replication defective mutant of HSV-1, which expresses a substantial level of viral genes without producing virus particles, is as efficient as wild-type HSV-1 in eliciting an HSV-specific cytotoxic T-lymphocyte (CTL) response. In this report, we have further evaluated the immunogenic potential of HSV-1-derived replication defective mutants by examining the generation of HSV-specific CTL following immunization with viruses that are severely restricted in viral gene expression due to mutations in one or more HSV alpha genes (ICP4, ICP27, ICP22, and ICP0). To measure the CTL responses induced by the HSV alpha-mutants, we have targeted two H-2Kb-restricted CTL epitopes: an epitope in a virion protein, gB (498-505), and an epitope in a nonvirion protein, ribonucleotide reductase (RR1 822-829). The HSV mutants used in this study are impaired in their ability to express gB while a majority of them still express RR1. Our findings demonstrate that a single immunization with these mutants is able to generate a strong CTL response not only to RR1 822-829, but also to gB498-505 despite their inability to express wild-type levels of gB. Furthermore, a single immunization with any individual mutant can also provide immune protection against HSV challenge. These results suggest that mutants which are restricted in gene expression may be used as effective immunogens in vivo.