Secretion from single pancreatic beta -cells was imaged using a novel technique in which Zn2+, costored in secretory granules with insulin, was detected by confocal fluorescence microscopy as it was released from the cells. Using this technique, it was observed that secretion from beta -cells was limited to an active region that comprised similar to 50% of the cell perimeter. Using ratiometric imaging with indo-1, localized increases in intracellular Ca2+ concentration ([Ca2+](i)) evoked by membrane depolarization were also observed. Using sequential measurements of secretion and [Ca2+](i) at single cells, colocalization of exocytotic release sites and Ca2+ entry was observed when cells were stimulated by glucose or K+. Treatment of cells with the Ca2+ ionophore 4-Br-A23187 induced large Ca2+ influx around the entire cell circumference. Despite the non-localized increase in [Ca2+](i), secretion evoked by 4-Br-A23187 was still localized to the same region as that evoked by secretagogues such as glucose. It is concluded that Ca2+ channels activated by depolarization are localized to specific membrane domains where exocytotic release also occurs; however, localized secretion is not exclusively regulated by localized increases in [Ca2+](i), but instead involves spatial localization of other components of the exocytotic machinery.