NK1.1+ T (NKT) cells are efficient regulators of early host responses which have been shown to play a role in tumor surveillance. The relevance of NKT cells in immune surveillance of viral infections, however, is not well understood. In this study, we investigated the functional relevance of NKT cells in controlling herpesvirus infections by using challenge with murine cytomegalovirus (MCMV) as the study model. This model has proven to be one of the best systems for evaluating the role of NK cells during virus infection. Using gene-targeted mice and alpha-galactosylceramide (alpha-GalCer) as an exogenous stimulator of NKT cells, we have analyzed the role of these cells in the immune surveillance of MCMV infection. Our studies in NKT-cell-deficient, T-cell receptor Jalpha281 gene-targeted mice have established that classical NKT cells do not play a critical role in the early clearance of MCMV infection. Importantly, however, activation of NKT cells by alpha-GalCer resulted in reduced viral replication in visceral organs. Depletion studies, coupled with analysis of gene-targeted mice lacking perforin and gamma interferon (IFN-gamma), have revealed that the antiviral effects of alpha-GalCer involve NK cells and have clearly demonstrated that the antiviral activity of alpha-GalCer, unlike the antitumor one, is critically dependent on both perforin and IFN-gamma.