Molecules that regulate encephalitogenic T cells are of interest for multiple sclerosis. In this study we show that protein kinase Cθ (PKCθ) is critical for the development of Ag-specific Th1 cells in experimental allergic encephalomyelitis (EAE), a model of multiple sclerosis. PKCθ-deficient mice immunized with myelin oligodendrocyte glycoprotein failed to develop cell infiltrates and Th1 cytokines in the CNS and were resistant to the development of clinical EAE. CD4 T cells became primed and accumulated in secondary lymphoid organs in the absence of PKCθ, but had severely diminished IFN-γ, TNF, and IL-17 production. Increasing Ag exposure and inflammatory conditions failed to induce EAE in PKCθ-deficient mice, showing a profound defect in the myelin oligodendrocyte glycoprotein-reactive T cell population. These data provide evidence of a pivotal role for PKCθ in the generation and effector function of autoimmune Th1 cells.