Protein kinase C-theta (PKC-theta) is essential for mature T cell activation; however, the mechanism by which it is recruited to the TCR signaling machinery is unknown. Here we show that T cell stimulation by antibodies or peptide-major histocompatibility complex (MHC) induces translocation of PKC-theta to membrane lipid rafts, which localize to the immunological synapse. Raft translocation was mediated by the PKC-theta regulatory domain and required Lck but not ZAP-70. In addition, PKC-theta was associated with Lck in the rafts. An isolated PKC-straight theta catalytic fragment did not partition into rafts or activate the transcription factor NF-kappa B, although addition of a Lck-derived raft-localization sequence restored these functions. Thus, physiological T cell activation translocates PKC-theta to rafts, which localize to the T cell synapse; this PKC-theta translocation is important for its function.