Class II histone deacetylases (HDACs) are signal-responsive repressors of gene transcription. In the heart, class II HDAC5 suppresses expression of genes that govern stress-induced cardiomyocyte growth. Signaling via pro-growth G protein coupled receptors triggers phosphorylation of HDAC5 on two serine residues (Ser(259) and Ser(498)), resulting in nuclear export of HDAC5 and de-repression downstream target genes. Although prior studies established a role for protein kinase D (PKD) in the regulation of HDAC5 phosphorylation, it remained unclear whether PKD functions directly or indirectly to control the phosphorylation status of this transcriptional repressor. Here, we demonstrate that PKD catalyzes direct phosphoryl-group transfer to Ser(498) of HDAC5. Each of the three PKD family members, PKD1, PKD2, and PKD3, is capable of phosphorylating HDAC5 (K(m) for substrate=2.07, 3.12, and 1.43microM, respectively), although PKD2 exhibits highest catalytic efficiency (k(cat)/K(m)=6.77min(-1)microM(-1)). Kinetic studies revealed that the three PKD isozymes phosphorylate HDAC5 through a random sequential mechanism, and that ATP has no effect on association of kinase with peptide substrate. In addition, we demonstrate that ADP competitively inhibits phosphorylation of HDAC5 (K(i)=8.50, 17.54, and 11.98microM for PKD1, PKD2, and PKD3, respectively). These findings define PKD as an HDAC kinase and thus suggest key roles for PKD family members in the control of chromatin structure and gene expression.