Protein kinase C delta (PKCdelta) is a Ser/Thr kinase which regulates numerous cellular processes, including proliferation, differentiation, migration and apoptosis. Here, we demonstrate that PKCdelta undergoes in vitro autophosphorylation at three sites within its V3 region (S299, S302, S304), each of which is unique to this PKC isoform and evolutionarily conserved. We demonstrate that S299 and S304 can be phosphorylated in mammalian cells following phorbol ester stimulation and that S299-phosphorylated PKCdelta is localised to both the plasma and nuclear membranes. These data indicate that PKCdelta is phosphorylated upon activation and that phospho-S299 represents a useful marker of the activated enzyme.