The Ca(2+) activated K(+) channel K(ca)3.1 is expressed in a variety of tissues. In the gastric gland it is expressed in the basolateral cell membrane. To determine the functional significance of K(ca)3.1 activity for gastric acid secretion, gastric acid secretion was determined in isolated glands from gene targeted mice lacking functional K(ca)3.1 (K(ca)3.1(-/-)) and from their wild type littermates (K(ca)3.1(+/+)). According to BCECF-fluorescence cytosolic pH in isolated gastric glands was similar in K(ca)3.1(-/-) and K(ca)3.1(+/+) mice. Na(ca)-independent pH recovery (ΔpH/min) following an ammonium pulse, a measure of H(ca)/K(ca) ATPase activity, was, however, significantly faster in K(ca)3.1(-/-) than in K(ca)3.1(+/+) mice. Accordingly, the luminal pH was significantly lower and the acid content significantly higher in K(ca)3.1(-/-) than in K(ca)3.1(+/+) mice. The abundance of mRNA encoding H(ca)/K(ca) ATPase and KCNQ1 was similar in both genotypes. Increase of extracellular K(ca) concentrations to 35 mM (replacing Na(ca)/NMDG) and treatment with histamine (100 μM) significantly increased ΔpH/min to a larger extent in K(ca)3.1(+/+) than in K(ca)3.1(-/-) mice and dissipated the differences between the genotypes. Carbachol (100 μM) increased ΔpH/min in both genotypes but did not abolish the difference between K(ca)3.1(-/-) and K(ca)3.1(+/+) mice. In K(ca)3.1(+/+) mice the K(ca)3.1 opener DCEBIO (100 μM) did not significantly alter basal ΔpH/min but significantly blunted ΔpH/min in the presence of carbachol. In conclusion, K(ca)3.1 activity suppresses carbachol stimulated gastric acid secretion.