Two regulatory systems are believed to control the contractile state of vascular smooth muscle in vitro. In one, crossbridges are phosphorylated by myosin light-chain (MLC) kinase; these phosphorylated crossbridges are believed to predominate during force development. In the other system, the crossbridges are unphosphorylated (latchbridges); the latchbridges are thought to prevail during force maintenance. The role of these systems in the control of vascular resistance in vivo is unknown. This study compared MLC phosphorylation with vascular tone in canine anterior tibial arteries in vitro and in situ. The arteries were frozen at various times before and during stimulation with norepinephrine. Under both conditions, the levels of MLC phosphorylation were low at rest and increased transiently during norepinephrine stimulation. This increase was associated with stress development in vitro and with an increase in vascular resistance in situ. Thus, the biochemical changes measured in arteries in situ parallel those measured in vitro suggesting that the in vitro models are appropriate and useful for studying the physiological function of blood vessels. To our knowledge, this is the first demonstration of changing levels of MLC phosphorylation in an artery in situ.