Natural killer (NK) cell sense virally infected cells and tumor cells through multiple cell surface receptors. Many NK cell–activating receptors signal through immunoreceptor tyrosine–based activation motif (ITAM)–containing adapters, which trigger both cytotoxicy and secretion of interferon-gamma (IFN-γ). Within the ITAM pathway, distinct signaling intermediates are variably involved in cytotoxicity and/or IFN-γ secretion. In this study, we have evaluated the role of protein kinase C-θ (PKC-θ) in NK-cell secretion of lytic mediators and IFN-γ. We found that engagement of NK-cell receptors that signal through ITAMs results in prompt activation of PKC-θ. Analyses of NK cells from PKC-θ–deficient mice indicated that PKC-θ is absolutely required for ITAM-mediated IFN-γ secretion, whereas it has no marked influence on the release of cytolytic mediators. Moreover, we found that PKC-θ deficiency preferentially impairs sustained extracellular-regulated kinase signaling as well as activation of c-Jun N-terminal kinase and the transcription factors AP-1 and NFAT but does not affect activation of NF-κB. These results indicate that NK cell–activating receptors require PKC-θ to generate sustained intracellular signals that reach the nucleus and promote transcriptional activation, ultimately inducing IFN-γ production.