The course of herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2) and varicella-zoster virus (VZV) infections in squamous epithelial cells cultured in a three-dimensional organotypic raft culture was tested. In these raft cultures, normal human keratinocytes isolated from neonatal foreskins grown at the air-liquid interface stratified and differentiated, reproducing a fully differentiated epithelium. Typical cytopathic changes identical to those found in the squamous epithelium in vivo, including ballooning and reticular degeneration with the formation of multinucleate cells, were observed throughout the raft following infection with HSV and VZV at different times after lifting the cultures to the air-liquid interface. For VZV, the aspects of the lesions depended on the stage of differentiation of the organotypic cultures. The activity of reference antiviral agents, acyclovir (ACV), penciclovir (PCV), brivudin (BVDU), foscarnet (PFA), and cidofovir (CDV), was evaluated against wild-type and thymidine kinase (TK) mutants of HSV and VZV in the raft cultures. ACV, PCV, and BVDU protected the epithelium against cytopathic effect induced by wild-type viruses in a concentration-dependent manner, while treatment with CDV and PFA proved protective against the cytodestructive effects induced by both TK+ and TK- strains. The quantification of the antiviral effects in the rafts were accomplished by measuring viral titers by plaque assay for HSV and by measuring viral DNA load by real-time PCR for VZV. A correlation between the degree of protection as determined by histological examination and viral quantification could be demonstrated The three-dimensional epithelial raft culture represents a novel model for the study of antiviral agents active against HSV and VZV. Since no animal model is available for the evaluation of antiviral agents against VZV, the organotypic cultures may be considered a model to evaluate the efficacy of new anti-VZV antivirals before clinical trials.