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-γ (PKC-γ) 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-γ. Analyses of NK cells from PKC-θ-deficient mice indicated that PKC-θ is absolutely required for ITAMmediated 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.