The pathogenicity of ribonucleotide reductase (RR)-null mutants (hrR3 and ICP6 delta) of herpes simplex virus (HSV) type 1 was studied after intracerebral and corneal inoculation in newborn and adult ICR mice. ICP6 delta failed to replicate in brains of mice greater than or equal to 8 days old but exhibited significant virulence for newborn mice as a result of viral replication in the brains. The RR- and a thymidine kinase (TK)-deficient mutant of HSV-1 strain KOS could grow in eye tissues of adult ICR mice. Viral DNA of hrR3 was detected in brain tissues of intracerebrally infected mice or in the trigeminal ganglia of corneally infected mice greater than or equal to 50 days after infection, and infectious hrR3 could be recovered from these tissues by superinfection of the mice with wild-type HSV-2. These observations indicate that pathogenicity of RR- mutants in mice is highly dependent on the physiologic state of tissues infected and that RR- mutants have the ability to establish latency in nervous system tissues of mice by either the peripheral or intracerebral route. It was also demonstrated that the inability of the RR- mutants to invade the central nervous system was efficiently complemented by simultaneous infection with another defective virus, the TK- mutant of KOS.