This study describes the outcome of herpes simplex virus type 1 (HSV-1) infection in an organotypic raft culture of spontaneously immortalized HaCat keratinocytes and human fibroblasts, as related to the virus load and epithelial stratification and differentiation. In this model, a confluent monolayer of HaCat keratinocytes was formed 60 h after seeding. Inoculation of HSV-1 before induction of differentiation by lifting of the culture to the air-liquid interface always resulted in a productive infection, but the virus yield was highest when the inoculation took place 72 h after seeding. Even at 0.1 p.f.u. per culture, the HaCat cultures became HSV positive. Infection of the full-thickness epithelium at 5 p.f.u. per culture resulted in a productive infection of the whole epithelium. The HaCat cells were about 10 times more sensitive to HSV-1 infection than the Vero cells in which the virus stocks were titrated. The raft cultures infected 30 min after lifting were negative by HSV-1 culture, and no HSV-1 antigen was detected by immunocytochemistry. PCR showed the presence of HSV-1 DNA and in situ hybridization showed reactivity with a latency-associated RNA probe, indicating the presence of a non-productive infection. Two different patterns of virus spread in epithelia were found: (i) lateral spread through the superficial layers of the epithelium and (ii) a demarcated infection throughout the whole thickness of the epithelium at the margins of the culture.