Protein kinase D (PKD)/protein kinase Cmicro (PKCmicro, a serine/threonine protein kinase with distinct structural and enzymological properties, is rapidly activated in intact cells via PKC. The amino-terminal region of PKD contains a cysteine-rich domain (CRD) that directly binds phorbol esters with a high affinity. Here, we show that treatment of transfected RBL 2H3 cells with phorbol 12,13-dibutyrate (PDB) induces a striking CRD-dependent translocation of PKD from the cytosol to the plasma membrane, as shown by real time visualization of a functional green fluorescent protein (GFP)-PKD fusion protein. A single amino acid substitution in the second cysteine-rich motif of PKD (P287G) prevented PDB-induced membrane translocation but did not affect PKD activation. Our results indicate that PKD translocation and activation are distinct processes that operate in parallel to regulate the activity and localization of this enzyme in intact cells.