Herpes simplex virus (HSV) is common throughout the world and is a target for vaccine development. Transcutaneous immunisation is a novel technique that uses the application of vaccine antigens in solution on the skin in the presence of cholera toxin (CT) as an adjuvant. This study investigated the potential of transcutaneous immunisation in C3H mice, using CT co-administered with whole inactivated HSV-1 (CT+HSVi) or HSV-1 antigens extracted from infected Vero cells (CT+HSVag) or a control protein (CT+BSA). The application of any of the three vaccines on to bare mouse skin resulted in the migration of Langerhans cells from the epidermis and in the production of serum antibodies to CT. Both HSV preparations generated serum and mucosal (faecal) antibodies to HSV, with the CT+HSVi vaccine being a more potent stimulator of humoral immunity. The CT+HSVag vaccine, however, was the more potent stimulator of cell-mediated immunity, giving rise to a strong delayed type hypersensitivity response and lymphocyte proliferation in vitro. When the mice were challenged by epidermal inoculation of HSV, the CT+HSVag vaccine induced a higher level of protection than the CT+HSVi vaccine, a result which may indicate that the efficacy of HSV vaccines depends on stimulation of cell-mediated rather than humoral responses. The success of topical vaccination suggests that the transcutaneous route may offer a promising potential for novel vaccine delivery which merits further investigation.