Abstract  Evidence from the pathology in cystic fibrosis (CF) and recent results in vitro indicate that HCO₃⁻ is required for gel‐forming mucins to form the mucus that protects epithelial surfaces. Mucus formation and release is a complex process that begins with an initial intracellular phase of synthesis, packaging and apical granule exocytosis that is followed by an extracellular phase of mucin swelling, transport and discharge into a lumen. Exactly where HCO₃⁻ becomes crucial in these processes is unknown, but we observed that in the presence of HCO₃⁻, stimulating dissected segments of native mouse intestine with 5‐hydroxytryptamine (5–HT) and prostaglandin E₂ (PGE₂) induced goblet cell exocytosis followed by normal mucin discharge in wild‐type (WT) intestines. CF intestines that inherently lack cystic fibrosis transmembrane conductance regulator (CFTR)‐dependent HCO₃⁻ secretion also demonstrated apparently normal goblet cell exocytosis, but in contrast, this was not followed by similar mucin discharge. Moreover, we found that even in the presence of HCO₃⁻, when WT intestines were stimulated only with a Ca²⁺‐mediated agonist (carbachol), exocytosis was followed by poor discharge as with CF intestines. However, when the Ca²⁺‐mediated agonist was combined with a cAMP‐mediated agonist (isoproterenol (isoprenaline) or vasoactive intestinal peptide) in the presence of HCO₃⁻ both normal exocytosis and normal discharge was observed. These results indicate that normal mucus formation requires concurrent activation of a Ca²⁺‐mediated exocytosis of mucin granules and an independent cAMP‐mediated, CFTR‐dependent, HCO₃⁻ secretion that appears to mainly enhance the extracellular phases of mucus excretion.