The gastric glands of the mammalian fundic mucosa are constituted by different cell types. Gastric fluid is a mixture of acid, alkali, ions, enzymes and mucins secreted by parietal, chief and mucous cells. We studied activation of acid secretion using Lysosensor Yellow-Blue in conjunction with fluo-3 to measure changes in pH and Ca²⁺ in isolated rabbit gastric glands. We evidenced a spatial heterogeneity in the amplitude of acid response along the gland axis under histamine and cholinergic stimulation. Carbachol induced a transitory pH increase before acidification. This relative alkalinization may be related to granule release from other cell types. Omeprazol inhibited the acid component but not the rise in pH. Histamine stimulated acid secretion without increase of lumen pH. We studied the relationship between Ca²⁺ release and/or entry and H⁺ secretion in glands stimulated by carbachol. Ca²⁺ release was associated with a fast and transient components of H⁺ secretion. We found a linear relationship between Ca²⁺ release and H⁺ secretion. Ca²⁺ entry was associated with a second slow and larger component of acid secretion. The fast component may be the result of activation of Cl^- and K⁺ channels and hence H⁺/K⁺ pumps already present in the membrane, whereas the slow component might be associated with translocation of H⁺//K⁺ pumps to the canaliculi. In conclusion, with cholinergic stimulation, gastric glands secrete a mixture of acid and other product(s) with a pH above 4.2, both triggered by Ca²⁺ release. Maintenance of acid secretion depends on Ca²⁺ entry and perhaps membrane fusion.