Contraction of smooth muscle involves myosin light chain (MLC) kinase catalyzed phosphorylation of the regulatory MLC, activation of myosin, and the development of force. However, this cannot account for all aspects of a smooth muscle contraction suggesting that other regulatory mechanisms exist. One potentially important technique to study alternate sites of contractile regulation is siRNA. The goal of this study was to determine if siRNA technology can decrease the levels of a specific protein and allow for the determination of how that protein effects contractile regulation. To achieve this goal we tested the hypothesis that casein kinase 2 (CK2) is part of the complex regulatory scheme present in vascular smooth muscle. Using intact strips of swine carotid artery, we determined that siRNA against CK2 produced a tissue that resulted in a ~60% knock-down after four days in organ culture. Intact strips of vascular tissue depleted of CK2 produced greater levels of force and exhibited an increased sensitivity to all stimuli tested. This was accompanied by an increase in crossbridge cycling rates but not by a change in MLC phosphorylation levels. Alpha toxin permeabilized vascular tissue depleted of CK2 also showed an increased sensitivity to calcium as compared to control tissues. Our results demonstrate that siRNA is a viable technique to study regulatory pathways in intact smooth muscle tissue. Our results also demonstrate that CK2 plays an important role in the mechanism(s) responsible for the development of force and crossbridge cycling by a MLC phosphorylation independent pathway.