Protein kinase C (PKC) is a family of serine/threonine kinases whose activity is controlled, in part, by phosphorylation on three conserved residues that are located on the catalytic domain of the enzyme, known as the activation-loop, the turn-motif, and the C-terminal hydrophobic-motif sites. Using a panel of phospho-specific antibodies, we have determined that PKC beta(I) and delta are constitutively phosphorylated on all three sites in unstimulated and activated T cells. Although PKC theta is constitutively phosphorylated at the activation-loop and turn-motif sites in T cells, PMA or anti-CD3/CD28 stimulation results in an increase in phosphorylation at the hydrophobic-motif (Ser695), an event that coincides with translocation of the enzyme from the cytosol/cytoskeleton to the membrane. Studies on the stimulus-induced phosphorylation of PKC theta demonstrate that an upstream kinase activity involving a conventional PKC isoform(s) and the PI3-kinase pathway, rather than autophosphorylation or the rapamycin-sensitive mTOR pathway, regulates this site in T lymphocytes. However, hydrophobic-motif phosphorylation does not appear to control membrane translocation, suggesting that this site may control other aspects of PKC theta signalling.