Caffeine is known to inhibit replication of herpes simplex virus (HSV)-1 and the therapeutic efficacy of caffeine (Cafon) gel has been shown in a mouse model cutaneously infected with HSV-1. In this study we examined the inhibitory effect of caffeine on infection with HSV-2 and acyclovir-resistant HSV-1 strains, thymidine kinase (TK)-deficient and phosphonoacetic acid (PAA)-resistant HSV-1 in vitro and in vivo. Caffeine inhibited plaque formation of HSV-2 and acyclovir-resistant HSV-1 strains and their EC50 values ranged from 0.42 to 1.11 mg/ml. Topical treatment with Cafon gel was significantly effective in retarding the development of skin lesions caused by cutaneous infection with HSV-2 and PAA-resistant HSV-1 and in reducing the virus yield of the skin infected with TK-deficient HSV-1. The results suggested that Cafon gel would be useful for the topical treatment of cutaneous infection with HSV-2 and acyclovir-resistant HSV strains.