We compared the penciclovir susceptibilities and pathogenesis phenotypes of mutants of Herpes simplex virus type 1 that are resistant to acyclovir and/or foscarnet. The mutants, which were derived from laboratory strain KOS, included six DNA polymerase mutants, a thymidine kinase negative mutant, a thymidine kinase partial mutant, and a double mutant. Two of four polymerase mutants not previously examined for penciclovir susceptibility exhibited modest resistance to this drug. A thymidine kinase negative mutant exhibited approximately 20-fold resistance while a thymidine kinase partial mutant was penciclovir-sensitive. Following intracerebral inoculation of 7-week old CD1 mice, the mutants ranged from exhibiting near wild-type neurovirulence (thymidine kinase partial) to modest attenuation (e.g. thymidine kinase negative) to more severe attenuation. Following corneal inoculation, three polymerase mutants exhibited modest deficits (relative to those of thymidine kinase negative mutants) in their abilities to replicate acutely in the ganglion and reactivate from latency. For mutant AraA(r)13, the deficit in ganglionic replication was shown to be due to its polymerase mutation by analysis of recombinant viruses derived by marker rescue. These results may have implications for issues of penciclovir action and resistance, for drug resistance in the clinic, and for the interactions of herpes viruses with the peripheral and central nervous systems.