The pH of the intestinal surface was measured in rat jejunum and ileum and in human jejunal biopsy samples by a 30 micron glass pH-electrode. This low surface pH became more neutral in the ileum and was less acid in the presence of 10 mM mucosal aminophylline or ouabain and in the absence of added 10 mM glucose; glucose was necessary for maintenance of the low surface pH over the incubation period. The surface pH values obtained under various conditions correlated with previously obtained estimates of the rate of acidification under similar conditions. Acidification was not a product of the submucosal layers but of structurally intact mucosal cells and unaccounted for by lactic and pyruvic acid appearance. The ouabain and aminophylline-inhibited serosal acidification seen in everted sacs was less than 2% of that occurring mucosally and might be accounted for solely by back-diffusion of hydrogen ion from the mucosal surface.