Protein kinase C-θ (PKCθ) is critical for TCR-initiated signaling in mature T cells, but initial reports found no requirement for PKCθ in thymocyte development. Thymocytes and peripheral T cells utilize many of the same signaling components and, given the significant role of PKCθ in peripheral T cells, it was surprising that it was not involved at all in TCR signaling in thymocytes. We decided to re-evaluate the role of PKCθ in thymocyte development using the well-characterized class II-restricted n3.L2 TCR-transgenic TCR model. Analysis of n3.L2 PKCθ-/- mice revealed a defect in thymocyte-positive selection, resulting in a 50% reduction in the generation of n3.L2 CD4 single-positive thymocytes and n3.L2 CD4 mature T cells. Competition between n3.L2 WT and n3.L2 PKCθ-/- thymocytes in bone marrow chimeras revealed a more dramatic defect, with a >80% reduction in generation of n3.L2 CD4 single-positive thymocytes derived from PKCθ-/- mice. Inefficient positive selection of n3.L2 PKCθ-/- CD4 single-positive cells resulted from "weaker" signaling through the TCR and correlated with diminished ERK activation. The defect in positive selection was not complete in the PKCθ-/- mice, most likely accounted for by compensation by other PKC isoforms not evident in peripheral cells. Similar decreased positive selection of both CD4 and CD8 single-positive thymocytes was also seen in nontransgenic PKCθ-/- mice. These findings now place PKCθ as a key signaling molecule in the positive selection of thymocytes as well as in the activation of mature T cells.