Gastric secretion of hydrochloric acid requires protons and chloride, yet the mechanisms and regulation of gastric chloride secretion remain unclear. We developed an in vivo technique to simultaneously measure acid/base and chloride secretion into the gastric lumen of anesthetized rats. The cannulated stomach lumen was perfused with weakly pH-buffered chloride-free solution containing a chloride-sensitive fluorophore [5 µM N-(ethoxycarbonylmethyl)-6-methoxyquinolinium bromide (MQAE)]. Gastric acid and chloride secretion was detected in gastric effluents by 1) flow-through pH electrode and 2) MQAE fluorescence. Gastric effluent was also collected at 1-min intervals for independent determination of chloride amount by chloridometer. In all conditions, both optical and chemical determinations of chloride report similar amounts of secreted chloride. During luminal perfusion with pH 5 solution, net acid and chloride secretion into the lumen was observed. Pentagastrin stimulated both secretions. In contrast, proton pump inhibition (omeprazole) caused alkalinization of the gastric effluent, but chloride secretion was not diminished. During luminal pH 3 perfusion, net alkali secretion was observed, and chloride secretion at luminal pH 3 was greater than pH 5. When tissue is pretreated with omeprazole at luminal pH 3, the addition of prostaglandin E2 synchronously stimulates both alkali and chloride secretion. Results suggest that both acid and alkali secretions are separately coupled with chloride secretion.