Contractile agonists can mobilize Ca2+from both intracellular and extracellular stores in smooth muscle. This study addresses the role of Ca2+mobilization as it relates to the complex manner by which Ca2+regulates the contractile system in smooth muscle. In swine carotid media, both histamine and phenylephrine produced initial rapid increases in myosin phosphorylation and stress. Stress was sustained for the duration of the stimulus while myosin phosphorylation slowly declined to steady-state levels. Removal of extracellular Ca2+or elimination of cellular Ca2+influx did not dramatically reduce the initial rapid increase in myosin phosphorylation produced by either agonist but reduced steady-state levels of myosin phosphorylation to basal values. Initial rapid increases in stress were seen, but stress was not maintained. Following depletion of Ca2+from sarcoplasmic reticulum, muscle activation by Ca2+influx in the presence of phenylephrine occurred without an initial transient in myosin phosphorylation, and stress developed slowly. Steady-state levels of myosin phosphorylation and stress were not different from control values. Similar results were obtained with histamine, although a small transient in myosin phosphorylation was also seen. These results are consistent with the hypothesis that the role of the sarcoplasmic reticulum in vascular smooth muscle is to provide high myoplasmic Ca2+concentrations causing extensive myosin phosphorylation and rapid crossbridge cycling leading to rapid stress development. In the presence of extracellular Ca2+, control levels of agonist-induced steady-state stress and myosin phosphorylation could be produced without an initial phosphorylation transient when intracellular Ca2+pools were depleted, suggesting that the sarcoplas mic reticulum is not required for the regulation of steady-state myoplasmic [Ca2+] during “latch.”