In order to determine the possible correlation of specific immune responses with protection against mortality and ocular disease following ocular herpes simplex virus type 1 (HSV-1) challenge, BALB/c mice were vaccinated with different doses and regimens of baculovirus-expressed gD. Neutralizing antibody, virus titres in the eyes, corneal scarring, and survival were measured. In addition, infiltration into the cornea of CD4+ T cells and cells containing the lymphokines interleukin-2 (IL-2), IL-4, IL-6 and tumour necrosis factor-alpha (TNF-alpha) were monitored on days 3, 7, 10, 14 and 21 post-challenge by immunocytochemistry. The vaccination regimens used induced varying degrees of immune responses and protection upon ocular challenge with HSV-1. Our results suggest that neutralizing antibody was the most important immune response in protecting mice against lethal ocular challenge and corneal scarring. TNF-alpha and IL-2 were not crucial in terms of survival and corneal scarring, since gD1 (one vaccination with 1 microg of gD) and gD0.1 (one vaccination with 0.1 microg of gD), both of which provided high levels of protection, showed no TNF-alpha or IL-2 expression. However, TNF-alpha and IL-2 were crucial in terms of virus clearance from the eyes, since gD3 (three vaccinations with 1 microg of gD), which had less virus in their eyes, had high numbers of TNF-alpha and IL-2 infiltrates. Finally, mock-vaccinated mice were not protected from death and corneal disease following HSV-1 challenge. Eyes of mock-vaccinated mice had little or no TNF-alpha or IL-2 responses and the strongest IL-4 and IL-6 responses.