DNAX accessory molecule-1 (DNAM-1) is a signal-transducing adhesion molecule involved in the cytolytic function mediated by CTL and NK cells. In the present study, we have investigated various perimeters of DNAM-1-mediated signaling and adhesion. Although adhesion of DNAM-1 to its ligand does not require divalent cations, protein synthesis, or RNA transcription, activation of protein kinase C (PKC) is required for DNAM-1 functioning. Furthermore, mutation of the putative PKC-binding site in the cytoplasmic domain of DNAM-1 (Ser 329 to Phe 329) prevents both ligand binding and PMA-induced phosphorylation of the DNAM-1 receptor. These results indicate that PKC phosphorylates Ser 329 of DNAM-1 and plays a critical role for both DNAM-1 adhesion and signaling.