The duodenal glands have been thought to play an important role in defense against proximal duodenal ulcer; however, the secretory mechanisms of these glands remain to be determined. In isolated duodenal acinar cells of the pig, we investigated the effects of ACh on intracellular Ca²⁺ concentration ([Ca²⁺]_i) and on membrane currents with fura 2 fluorometry and the patch clamp technique. ACh caused a transient increase in [Ca²⁺]_i, and the increase was markedly inhibited by atropine or 4-diphenylacetoxy-N-methylpiperidine methiodide but not by hexamethonium, pirenzepine, or methoctramine. The expression of mRNA for the M₃ subtype far exceeded that for either M₁ or M₂ as revealed by real-time quantitative PCR and in situ hybridization. The rise in [Ca²⁺]_i evoked by ACh was largely inhibited by thapsigargin but slightly affected by extracellular Ca²⁺ deprivation. Caffeine had no effect on [Ca²⁺]_i. ACh elicited Ca²⁺-dependent K⁺ currents, a finding similar to the response to inositol 1,4,5,-trisphosphate applied intracellularly. These results suggest the presence of M₃ receptors linked to Ca²⁺ release in porcine duodenal glands.