Antibodies against MUC2, MUC3, and MUC5AC peptide epitopes stained the secretory contents of all goblet cells in the human colon-derived HT29-18N2 cell line. In contrast, four carbohydrate-specific monoclonal antibodies stained mucin glycoforms in consistent subsets of goblet cells. Cholinergic agonist-evoked decreases in total mucin stores were not always mirrored by proportional changes in mucin glycoforms in the same monolayers. Selective secretion of mucin glycoforms did not result from differences in receptor distribution, since cholinergic stimulation was found to increase intracellular free calcium in all cells and selective secretion was also observed when the cells were directly stimulated with the protein kinase C activator phorbol myristate acetate. The results demonstrate that goblet cells cycle through transient periods in which their exocytotic response is unresponsive to cholinergic or protein kinase C-mediated stimuli. Goblet cells replenished intracellular mucin stores to control levels within 1 h, but the relative proportion of mucin glycoforms was not always restored until 24 h after stimulation.