The ultrastructure and function of the cytoskeleton in intestinal goblet cells was investigated in colonic mucosa from rabbits and monkeys. This exocrine cell is unusual in that its secretory granules are stored as a compact apical mass limited by a dense, cup-shaped layer of cytoplasm called the "theca." Ultrastructural analysis of this cytoplasmic layer in rabbit goblet cells permeabilized with Triton X-100, treated with S4 fragments of heavy meromyosin, and fixed in the presence of tannic acid revealed that it contains an orderly arrangement of microtubules and intermediate filaments, but no detectable actin filaments. Microtubules are arranged vertically, like barrel staves, along the inner aspect of the theca. Intermediate filaments are arranged in two contiguous layers: an inner, basket-like network and an outer series of circumferential bundles resembling the hoops of a barrel. Autoradiography of [3H]glucosamine-labeled human and rabbit cells maintained in organ culture without secretagogues had provided preliminary data suggesting that labeled secretory granules migrate preferentially along the periphery of the apical granule mass, adjacent to the theca, toward the luminal cell surface. In this study, we confirm this observation and show that colchicine inhibits this movement. Cholinergic secretagogues induce rapid release of mucin by compound exocytosis of the granules stored in the theca. This secretory activity is not inhibited by colchicine, presumably because it does not require granule movement. The cuplike shape of the theca is unaltered during rapid release of stored granules. Because the shape is unaltered after 6 h of colchicine treatment, it appears to be maintained by the intermediate filament layers.