Recent studies have documented direct interactions between 14-3-3 proteins and several oncogene and proto-oncogene products involved in signal transduction pathways. Studies on the effects of 14-3-3 proteins on protein kinase C (PKC) activity in vitro have reported conflicting results, and previous attempts to demon¬strate a direct association between PKC and 14-3-3 were unsuccessful. Here, we examined potential physical and functional interactions between PKCθ, a Ca²⁺-independent PKC enzyme which is expressed selectively in T lymphocytes, and the 14-3-3Τ isoform in vitro and in intact T cells. PKCθ and 14-3-3τ coimmunoprecipitated from Jurkat T cells, and recombinant 14-3-3τ interacted directly with purified PKCθ in vitro. Transient overexpression of 14-3-3τ suppressed stimulation of the interleukin 2 (IL-2) promoter mediated by cotrans-fected wild-type or constitutively active PKCθ, as well as by endogenous PKC in ionomycin- and/or phorbol ester-stimulated cells. This did not represent a general inhibition of activation events, since PKC-independent (but Ca²⁺-dependent) activation of an IL-4 promoter element was not inhibited by 14-3-3τ under similar conditions. Overexpression of wild-type 14-3-3τ also inhibited phorbol ester-induced PKCθ translocation from the cytosol to the membrane in Jurkat cells, while a membrane-targeted form of 14-3-3τ caused increased localization of PKCθ in the particulate fraction in unstimulated cells. Membrane-targeted 14-3-3τ was more effective than wild-type 14-3-3τ in suppressing PKCθ-dependent IL-2 promoter activity, suggesting that 14-3-3τ inhibits the function of PKCθ not only by preventing its translocation to the membrane but also by associating with it. The interaction between 14-3-3 and PKCθ may represent an important general mechanism for regulating PKC-dependent signals and, more specifically, PKCθ-mediated functions during T-cell activation.