We have constructed and characterized a mutant of herpes simplex virus type 2 (HSV-2) which was inserted a modified lacZ gene, placed under the control of HSV-1 beta 8 promotor, into the US3 protein kinase gene. The mutant, L1BR1, could not induce the virus-encoded protein kinase activity, but could replicate in Vero cells as efficiently as the parental virus. When the biological properties of L1BR1 were examined in mice by using four routes (footpad, intraperitoneal, corneal, and intracerebral) of infection, the mutant displayed the route-dependent reduction of virulence; after inoculation by footpad and intraperitoneal routes, the mutant was more than 10,000-fold less virulent than the parental virus, but it exhibited only about a 10-fold decrease in virulence following the corneal and intracerebral infection. In the intraperitoneal inoculation into adult mice, the replication of L1BR1 in the liver and spleen was severely restricted, but in newborn mice the mutant could grow as well as the parental virus in these organs. The adoptive transfer of peritoneal macrophages from adult mice resulted in a marked inhibition in the replication of L1BR1 in the liver and spleen of newborn mice, while the transfer exhibited little or no effect on the production of the wild-type virus in these organs. We also found that the mutant, unlike the parental virus, could not replicate in precultured peritoneal macrophages from adult mice. Taking these observations together, it seems likely that L1BR1 lost the ability to overcome the mononuclear-phagocytic defense system and thereby lost its pathogenicity by intraperitoneal and footpad routes. Furthermore, the mutant was shown to be rescued by a 4.8-kb HindIII/Xbal fragment containing the entire US3 open reading frame. However, we could not rule out the possibility that some of the phenotypes of L1BR1 are due to mutations in the US3-neighboring genes, US2 and US4.