Summary2B4 (CD244) is a member of the CD2 subset of the immunoglobulin superfamily and functions as a triggering molecule on natural killer (NK) cells. Previously, we have found that 2B4‐mediated activation of NK cells involves complex interactions involving LAT, Ras, Raf, ERK and p38 and that cytolytic function and cytokine production may be regulated by distinct pathways. Here we assessed the role of protein kinase C (PKC) in 2B4‐mediated cytotoxicity of YT cells, a human NK cell line. Our data indicate that PKC‐δ is activated upon stimulation with monoclonal antibody against 2B4. Treatment with the PKC inhibitor, bisindolylmaleimide I (Gö6850), of YT cells or YT cells depleted of Ca2+‐dependent isoforms of PKC prior to 2B4 stimulation, resulted in inhibition of natural cytotoxicity and redirected antibody‐dependent cellular cytotoxicity. However, inhibition of PKC failed to block 2B4 stimulation of interferon‐γ secretion as opposed to pretreatment with LY294002, a phosphoinositide 3‐kinase inhibitor. We also examined the effect of phorbol 12‐myristate 13‐acetate (PMA) induction on 2B4 gene transcription. PMA induction resulted in a more than two‐fold increase of 2B4 transcription. However, when we introduced a three‐base substitution mutation to disrupt the activator protein‐1 binding site at (−106 to −100) in the 2B4 promoter, we found complete loss of transcriptional activity, including the two‐fold increase due to PMA induction of PKC. The present study indicated that PKC may play an important role in 2B4 signalling and activator protein‐1 activation.