NKT cells are specialized cells of the immune system that bear both T cell and NK cell markers. Classical NKT cells display TCRs of restricted heterogeneity (Vα14-Jα281) and recognize lipid antigens (e.g., α-galactosyl ceramide) presented by nonpolymorphic CD1 molecules. Recently, other nonclassical NKT subsets have been recognized, including NKT cells not reactive with CD1d-α-galactosyl ceramide complexes. The biological functions of these cells are unknown. Here, we show that nonclassical NKT cells that are CD1d restricted but nonreactive to α-GalCer are activated in response to hepatocytes expressing hepatitis B viral antigens in a transgenic mouse model of acute hepatitis B virus infection. Our results document the first in vivo function for such nonclassical NKT cells and suggest a role for these cells in the host response to HBV infection.