We investigated the organization of spontaneous rises in cytosolic free Ca2+ concentration ([Ca2+]i) due to electrical activity in acute pituitary slices. Real time confocal imaging revealed that 73% of the cells generated fast peaking spontaneous [Ca2+]i transients. Strikingly, groups of apposing cells enhanced their [Ca2+]i in synchrony with a speed of coactivation >1,000 microm/s. Single-cell injection of Neurobiotin or Lucifer yellow labeled clusters of cells, which corresponded to coactive cells. Halothane, a gap junction blocker, markedly reduced the spread of tracers. Coupling between excitable cells was mainly homologous in nature, with a prevalence of growth hormone-containing cells. We conclude that spontaneously active endocrine cells are either single units or arranged in synchronized gap junction-coupled assemblies scattered throughout the anterior pituitary gland. Synchrony between spontaneously excitable cells may help shape the patterns of basal secretion.