Protein kinase C-theta (PKCtheta) was initially isolated as an important PKC isoform expressed in T cells, although its expression is not restricted to these cells. Despite the central function of PKCtheta in several immune responses, its role in the antitumor response against MHC class I (MHC-I)-negative cells has not been investigated. This is an important issue because most tumor cells growing in vivo down-regulate MHC-I expression to escape the CTL-mediated response. In the present work, we show that in vivo development of a MHC-I-deficient tumor (RMA-S) is much favored in PKCtheta(-/-) mice compared with wild-type mice. This is associated with a reduced recruitment of NK cells to the site of tumor development and a reduced activation status of recruited NK cells. This correlates with a reduced ex vivo and in vivo cytotoxic potential of NK cells isolated from PKCtheta(-/-) mice treated with polyinosinic:polycytidylic acid. Consistently, polinosinic:cytidilic acid treatment induces PKCtheta expression and activation of its enzymatic activity in NK cells in an indirect manner. These observations underline the relevance of PKCtheta as a key molecule in NK cell-mediated antitumor immune surveillance.