The mammalian esophagus has the capacity to secrete a bicarbonate and mucin rich fluid into the esophageal lumen. These secretions originate from the submucosal glands (SMG) and can contribute to esophageal protection against refluxed gastric acid. The cellular mechanisms by which glandular cells achieve these secretions are largely unknown. To study this phenomenon, we used the pH-stat technique to measure luminal alkali secretion in an isolated, perfused pig esophagus preparation. Immunohistochemistry was used to localize receptors and transporters involved in HCO₃ ^- transport. The SMG-bearing esophagus was found to have significant basal alkali secretion, predominantly HCO₃ ^-, which averaged 0.21±0.04 µEq/hour.cm². This basal secretion was doubled when stimulated by carbachol but abolished by HCO₃ ^- or Cl^- removal. Basal and carbachol stimulated secretions were also blocked by serosal application of atropine, pirenzipine, DIDS, methazolamide (MTZ) and ethoxzolamide (ETXZ). The membrane-impermeable CA inhibitor benzolamide (BNZ), applied to the serosal bath, partially inhibited basal HCO₃ ^- secretion and blocked the stimulation by carbachol. Immunohistochemistry using antibodies to M₁ cholinergic receptor or carbonic anhydrase-II enzyme showed intense labeling of duct cells and serous demi-lunes but no labeling of mucous cells. Labeling with an antibody to Na⁺-(HCO₃ ^-)_n (rkNBC) was positive in ducts and serous cells while labeling for Cl^-/HCO₃ ^- exchanger (AE2) was positive in duct cells but less pronounced in serous cells. These data indicate that duct cells and serous demi-lunes of SMG play a role in HCO₃ ^- secretion, a process that involves M₁ cholinergic receptor stimulation. HCO₃ ^- transport in these cells is dependent on cytosolic and serosal membrane-bound carbonic anhydrase. HCO₃ ^- secretion is also dependent on serosal Cl^- and is mediated by DIDS-sensitive transporters, possibly NBC and AE2.