Clinical studies and in vitro data from isolated parietal cells suggest that acute Helicobacter pylori infection inhibits acid secretion. Gastric acidification is mediated by H+-K+-ATPase, an integral protein of parietal cell apical membranes. To test the hypothesis that H. pyloridownregulates H+-K+-ATPase α-subunit (HKα) gene expression and to identify potential intracellular signaling pathways mediating such regulation, we transfected human gastric adenocarcinoma (AGS) cells with human and rat HKα 5′-flanking DNA fused to a luciferase reporter plasmid. Histamine caused dose-dependent, cimetidine-sensitive (10−4M) increases in cAMP, free intracellular Ca2+, and HKα promoter activation in AGS cells. H. pylori infection of transfected AGS cells dose dependently inhibited basal and histamine-stimulated HKα promoter activity by 80% and 66%, respectively. H. pylori dose dependently inhibited phorbol myristate acetate-induced (10−7M) and staurosporine- (10−7M) and calphostin C-sensitive (5 × 10−8M) activation of HKα promoter. Also, H. pylori inhibited epidermal growth factor (EGF) (10−8M), genistein-sensitive (5 × 10−5M) activation of HKα promoter, reducing activity to 60% of basal level. These data suggest that H. pyloriinhibits HKα gene expression via intracellular pathways involving protein kinases A and C and protein tyrosine kinase, AGS cells have functional histamine H2and EGF receptors, and transiently transfected AGS cells are a useful model for studying regulation of HKα gene expression.