Protein kinase D (PKD) is a novel serine/threonine kinase structurally distinct from all protein kinase C (PKC) isoforms but which like classic and novel PKCs is activated by phorbol esters and diacylglycerol. This study investigated the regulation of PKD in vascular smooth muscle cells (VSMC) by physiological regulators of VSMC function and growth factors. Treatment of rabbit aortic VSMC with phorbol ester, angiotensin II and PDGF-BB all stimulated PKD activity in a time- and concentration-dependent manner in VSMC. The effect of angiotensin II was particularly rapid and potent (maximum stimulation within 1 min and at 0.5 nM). In contrast, the maximum effect of PDGF-BB was obtained after 5 min. Other factors, including basic FGF, IGF-I, IGF-II, endothelin-1 and endothelin-2, had no effect on PKD activity in VSMC. These results show for the first time that PKD activity is regulated in VSMC, and is activated by the vasoconstrictor angiotensin II. PKD may be an important mediator for the biological function(s) of one or more PKC isoforms in VSMC and/or may represent a component of a novel PKC-independent signalling pathway in VSMC.