Protein kinase Cη (PKCη) is highly abundant in T cells and is recruited to the immunological synapse that is formed between a T cell and a cognate antigen-presenting cell; however, its function in T cells is unknown. Here, we showed that PKCη was required for the activation of mature CD8+ T cells by stimulation through the T cell receptor. PKCη−/− T cells showed poor proliferation in response to stimulation by antigen as compared to wild-type T cells, a trait shared with T cells deficient in PKCθ, the most abundant PKC isoform in T cells, and the only PKC previously thought to have a specific role in T cell activation. In contrast, defective homeostatic proliferation, a function requiring recognition of self antigens, was only observed in PKCη- deficient T cells. PKCη was dispensable for the development of thymocytes; however, thymocytes from mice doubly deficient in PKCη and PKCθ exhibited poor positive selection, indicating some redundancy between the PKC isoforms. PKCη and PKCθ had opposing effects on relative numbers of CD4+ and CD8+ T cells, because PKCη−/− mice had a higher ratio of CD4+ to CD8+ T cells compared to that of wild-type mice, whereas PKCθ−/− mice had a lower ratio. In mice deficient in both PKC isoforms, the ratio of CD4+ to CD8+ T cells returned to normal. Together, these data suggest that whereas PKCη shares redundant roles with PKCθ in T cell biology, it also performs nonredundant functions that are important for homeostasis and activation of T cells.