A herpes simplex virus type 2 (HSV-2) mutant deleted in the PK domain of the large subunit of ribonucleotide reductase (ICP10) was evaluated as a potential vaccine for the prevention of HSV-2 infection and disease. This virus, designated ICP10ΔPK, expressed a 95 kDa ICP10 protein that lacked PK activity and transforming potential. ICP10ΔPK was growth compromised in dividing and nondividing cells in culture. In dividing cells, onset of virus growth was delayed, with replication initiating at 10–15 h p.i. depending on the multiplicity of infection. In addition to the delayed growth onset, virus replication was significantly impaired (1000-fold lower titers) in nondividing cells. A revertant virus (HSV-2(R)) expressed ICP10, regained transforming activity and had wild type growth properties. ICP10ΔPK was growth compromised also in infected animals. It was isolated from the site of infection on day 2, but not day 7 p.i. and its titers at this time (2×10² pfu/ml) were significantly lower than those of HSV-2 (5×10⁴ pfu/ml). Mice given high titers of ICP10ΔPK (5×10⁷ pfu/footpad) remained free of clinical symptoms and survived infection during a 21-day follow-up period and virus was not isolated from latently infected ganglia at 30 days p.i. ICP10ΔPK immunized animals developed HSV-specific humoral and T-cell responses and evidenced absolute protection from HSV-2 infection and virus-induced disease.