We have examined the growth of three epitheliotropic viruses in cultures of human epidermal keratinocytes: herpes simplex virus (HSV) type 1, adenovirus type 2 (Ad-2), and human papillomavirus (HPV) type 1. Differences were noted in the level of expression of each virus, and these differences may be related to a dependency or lack of dependency on keratinocyte differentiation for complete viral growth. Of the three viruses studied, HSV was the only one to replicate productively in all cells of the culture. Its expression was independent of keratinocyte differentiation. This is unlike Ad-2, which infected all cells in the culture but replicated productively only in the suprabasal cells. Basal keratinocytes were shown to be infected, but for unknown reasons, they appeared in most instances to be nonpermissive for Ad-2 replication. Infected basal keratinocytes became permissive when they reached a suprabasal position. Ad-2 appears to require keratinocyte differentiation for full expression in culture. Following infection with HPV, cultured keratinocytes showed no evidence of productive replication. However, 50 to 250 copies of HPV DNA could be detected in each cell (average) as stable nonintegrated molecules. Viral DNA replication has been shown to occur in the younger cells and not in the older, more differentiated keratinocytes. The failure of HPV to be fully expressed in culture may be related, in part, to incomplete differentiation of the keratinocyte in vitro. The major conclusions of this study are (1) that keratinocyte differentiation is likely to play a role in the expression of some epitheliotropic viruses in culture, and (2) that keratinocyte differentiation may be a factor in the pathogenesis of certain viral diseases of keratinizing epithelia.