According to previous observations, basal gastric acid secretion is downregulated by phosphoinositol-3-(PI3)-kinase, phosphoinositide-dependent kinase (PDK1) and protein kinase B (PKBß/Akt) signaling. PKB/Akt phosphorylates glycogen synthase kinase GSK3. The present study explored whether PKB/Akt-dependent GSK3-phosphorylation modifies gastric acid secretion. UtilizingBCECF-fluorescence, basal gastric acid secretion was determined from Na⁺-independent pH-recovery (?pH/min) following an ammonium pulse, which reflects H⁺/K⁺-ATPase activity. Experiments were performed in gastric glands from gene targeted mice (gsk3^KI) with PKB/SGK insensitive GSKa,b, in which the serines within the PKB/SGK phosphorylation site were replaced by alanine (GSK3a^21A/21A, GSK3ß^9A/9A). As a result, the cytosolic pH in isolated gastric glands was similar in gsk3^KI and their wild type littermates (gsk3^WT). However, ?pH/min was significantly larger in gsk3^KI than in gsk3^WT mice. ?pH/min was virtually abolished by H⁺/K⁺-ATPase inhibitor omeprazole (100mM) in gastric glands from both gsk3^KI and gsk3^WT. Plasma gastrin levels were lower in gsk3^KI than in gsk3^WT. Both, increase of extracellular K⁺ concentration to 35mM (replacing Na⁺/NMDG) and treatment with forskolin (5mM), significantly increased ?pH/min to virtually the same value in both genotypes. Protein kinase A (PKA) inhibitor H89 (150nM) and H₂-receptor antagonist ranitidine (100mM) decreased ?pH/min in gsk3^KI but not gsk3^WT and again abrogated the differences between the genotypes. The protein abundance of phosphorylated but not of total PKA was significantly larger in gsk3^KI than in gsk3^WT. In conclusion, basal gastric acid secretion is enhanced by disruption of PKB/SGK-dependent phosphorylation and inhibition of GSK3. Thus, inhibition of GSK3 participates in the signaling of PI3-kinase-dependent downregulation of basal gastric acid secretion.