The importance of activation loop phosphorylation in the regulation of protein kinase D (PKD/protein kinase C (PKC) μ) activity has become controversial. In order to clarify the mechanism(s) of PKD activation, we developed a novel phosphospecific antibody recognizing phosphorylated Ser 748 in PKD (pS748). Western blot analysis with the pS748 antibody, carried out with a variety of PKD forms and in a variety of cell types including full-length PKD transfected in COS-7 and HEK 293 cells, a green fluorescent protein-PKD fusion protein transfected in either Swiss 3T3 fibroblasts or Madin-Darby canine kidney epithelial cells, and endogenous PKD expressed in A20 lymphocytes and Rat-1 fibroblasts, indicated that Ser 748 phosphorylation was absent from unstimulated cells. In contrast, dramatic increases in Ser748 phosphorylation were induced by phorbol esters, bombesin, or cross-linking of B lymphocyte antigen receptors or by cotransfection with active PKCε of PKCη. Western analysis using a second phosphospecific antibody, which primarily recognizes PKD phosphorylated at Ser744, revealed that Ser744 phosphorylation accompanies Ser748 phosphorylation during PKD activation in vivo. Ser744/Ser748 phosphorylation requires PKC but not PKD activity, indicative of transphosphorylation. Our results provide new experimental evidence indicating that activation loop phosphorylation at Ser744 and Ser748 occurs during PKD activation in vivo and support the notion of a PKC-PKD phosphorylation cascade.