A rise in cytosolic free Ca2+is the immediate trigger for contraction in mammalian vascular smooth muscle. We used the fluorescent calcium indicator fura-2 and digital imaging microscopy to study the spatial distribution of intracellular Ca2+in arterial myocytes and the changes elicited by activation with norepinephrine (NE). Viable arterial myocytes were obtained from bovine tail arteries by enzymatic digestion. In modified Krebs' solution containing 1.8 mM Ca2+, these myocytes were relaxed and spindle-shaped. The cells contracted rapidly when exposed to NE or high-K+solution ejected from a micropipette; they relaxed slowly when the activator was washed away. NE evoked a rise hi Ca2+concentration ([Ca2+]) in the cells within 100 msec, at a time when the cells had not yet begun to contract. Maximal [Ca2+] levels were attained within 600 msec, at which time the cells were substantially contracted. Digital analysis of images of cellular fura-2 fluorescence revealed that the intracellular [Ca2+] was relatively uniformly distributed prior to activation, with an average resting level of 111±14 nM (n=6). During NE-evoked contractions, intracellular [Ca2+] increased, and the distribution of [Ca2+] became much more heterogeneous. On recovery from activation, the cells relaxed, usually attaining >90percent; of their original resting length. In contrast to the relatively uniform Ca2+distribution observed prior to NE activation, discrete regions of elevated [Ca2+] were observed throughout the recovered cells. The large spatial variation of [Ca2+] after cell activation implies that Ca was sequestered at localized sites in the cell during relaxation.