Penciclovir has potent antiviral activity against varicella-zoster virus (VZV). We have characterized the inhibitory effects of penciclovir and acyclovir on the plaque formation of cell-free VZV and cross-resistance of acyclovir-resistant VZV to penciclovir. The apparent effective concentration for 50% plaque reduction (EC50) of penciclovir determined on the third day was significantly lower than that determined on the fourth or fifth day. The size of plaques was smaller in the presence of penciclovir than in the presence of acyclovir. The effective concentrations for 50% reduction of the number of infected cells per plaque were 1.40 and 5.00 micrograms/ml for penciclovir and acyclovir, respectively. Thus penciclovir suppressed spread of infection within developing plaques more efficiently than acyclovir. Five acyclovir-resistant VZV strains with altered DNA polymerase selected by acyclovir were examined for cross-resistance to penciclovir. They were 11- to 18-fold more resistant to ACV than the parent strain, but only 4- to 5-fold more resistant to PCV. Penciclovir-triphosphate carrying the 3'-hydroxyl group of 2'-deoxyribose might have better affinity to the altered viral DNA polymerase than acyclovir-triphosphate without the 3'-hydroxyl group.