Cytokines can be used as adjuvants to enhance and direct protective immune responses induced by vaccines. IL-12, a cytokine that favors the maturation of Th1-type cells and stimulates associated cell-mediated responses was evaluated as immunologic adjuvant for a viral vaccine in a mouse challenge model. When it was administered together with inactivated pseudorabies virus, a herpes simplex virus related alpha-herpesvirus, increased production of IFN-gamma by ex vivo-stimulated splenocytes was observed as well as augmented production of antiviral serum lgG2a. This was associated with increased protection against a lethal challenge infection. Infection of IfN-gamma-neutralizing Ab reduced the increased antiviral resistance in IL-12-treated mice. Also, in mice bearing an inactivated IFN-gamma-receptor gene IL-12 failed to stimulate protection against challenge and the synthesis of antiviral lgG2a. However, in these IFN-gamma-receptor knockout mice, increased antiviral lgG2b levels and enhanced IFN-gamma-secretion, with minimal IL-4 production, by ex vivo-stimulated splenocytes was observed. In wild-type mice administration of recombinant IFN-gamma but not IL-2 mimicked the immune-stimulating activity of IL-12; it is therefore likely that the IL-12 adjuvant activity is largely mediated by physiologic IFN-gamma.