The opossum esophagus, like that of humans, contains a network of submucosal glands with the capacity to secrete bicarbonate ions into the esophageal lumen. To evaluate the role of these glands in protecting the epithelial surface from acid insult, we measured the lumen-to-surface pH gradient in opossum esophagus at different luminal pH and compared it to that of rabbit esophagus, an organ devoid of submucosal glands. Sections of opossum and rabbit esophageal epithelium were mounted luminal side up in a modified Ussing chamber. pH-sensitive microelectrodes, positioned within 5 µm of the epithelial cell surface, were used to monitor surface pH during perfusion with solutions of different pH. At luminal pH 7.5, the pH_s of both opossum and rabbit were similar (pH_s = 7.5). Lowering luminal pH from 7.5 to 3.5 in opossum decreased pH_s to 4.2 ± 0.16, a value significantly higher than pH of perfusate, whereas in rabbit this maneuver decreased pH_s to 3.69 ± 0.08, a value not significantly different from pH of perfusate. In opossum but not in rabbit, addition of carbachol to the serosal solution increased basal pH_s to 7.8 ± 0.1 and significantly blunted the decline in pH_s on perfusion with acidic Ringer solution (pH 3.5), with pH_s falling to 5.6 ± 0.45. The effect of carbachol on surface buffering was inhibited by prior treatment with atropine. Luminal acidification to pH 2.0 in opossum (as in rabbit) abolished the lumen-to-surface pH gradient even after addition of serosal carbachol. We conclude that the presence of submucosal glands in esophagus contributes through bicarbonate secretion to creation of a lumen-to-surface pH gradient. Although this gradient can be modulated by carbachol, its capacity to buffer (and therefore to protect) the epithelial surface against back-diffusing H⁺ is limited and dissipated at pH 2.0.