We have recently shown that myristoylated alanine-rich C-Kinase (MARCKS), a membrane-bound phosphatidylinositol 4,5-bisphosphate (PIP2) binding protein (1), is expressed in vascular smooth muscle cells (VSMCs) and is involved in regulating ion channels linking to controlling vascular contractility (2). Therefore the present work investigates the functional role of MARCKS in regulating vascular tone. Male wistar rats (8-12 weeks of age) were killed by cervical dislocation in accordance with Schedule 1 of the UK Scientific Procedures Act, 1986. Isometric tension recordings were conducted on segments of second order branches of the mesenteric artery. Vessel segments were mounted on a wire myograph, normalised, and set to a resting tension of about 2mN. Bath application of MANS peptide (100nM-100µM), a cell-permeable MARCKS inhibitor, evoked concentration-dependent increases in resting tension with a mean EC50 valve of about 30µM. 100µM MANS peptide evoked vasoconstrictions with a similar amplitude to those responses induced by 10µM methoxamine. Importantly, co-application of 3µM nicardipine abolished MANS peptide-induced contractions. To investigate if MARCKS regulates vascular contractility by altering VGCC activity, we studied the effect of 100µM MANS peptide on whole-cell voltage-gated channel (VGCC) activity in freshly isolated rat mesenteric artery VSMCs. VGCC activity was evoked by applying 300ms voltage steps from -80mV to +40mV at 10mV intervals from a holding potential of -60mV. Bath application of 100µM MANS peptide significantly shifted the activation curve of VGCC activity to left, with the activation threshold shifting from about -30mV to -60mV, and peak amplitude increasing from about 3pA/pF at +20mV to 5pA/pF at -10mV. Pre-treatment of VSMCs with 20µM Wortmannin, a PI4 kinase inhibitor which depletes PIP2 levels, abolished the excitatory effects of 100µM MANS peptide on whole-cell VGCC activity. This study is the first study to demonstrate that MARCKS modulates vascular reactivity. Moreover it indicates that MARCKS controls vascular tone by regulating VGCC activity through a PIP2-dependent process.