Drebrins are highly expressed in brain where they may regulate actin filament formation in dendritic spines. Recently, the drebrin E2 isoform was detected in certain epithelial cell types including the gastric parietal cell. In gastric parietal cells, activation of HCl secretion is correlated with actin filament formation and elongation within intracellular canaliculi, which are the sites of acid secretion. The aim of this study was to define the pattern of drebrin expression in gland units in the intact rabbit oxyntic gastric mucosa and to initiate approaches to define the functions of this protein in parietal cells. Drebrin E2 expression was limited entirely or almost entirely to parietal cells and depended upon the localization of parietal cells along the gland axis. Rabbit drebrin E2 was cloned and found to share 86% identity with human drebrin 1a and to possess a number of cross-species conserved proteinprotein interaction and phosphorylation consensus sites. 2D Western blot and phosphoaffinity column analyses confirmed that drebrin is phosphorylated in parietal cells and several candidate phosphorylation sites were identified by mass spectrometry. Overexpression of epitope-tagged drebrin E2 led to the formation of microspikes and F-actin rich ring-like structures in cultured parietal cells and suppressed cAMP-dependent acid secretory responses. In MDCK cells, coexpression of epitope-tagged drebrin and the Rho family GTPase, Cdc42, which induces filopodial extension, produced an additive increase in the length of microspike projections. Coexpression of dominant-negative Cdc42 with drebrin E2 did not prevent drebrin-induced microspike formation. Taken together, these findings suggest that 1) drebrin can induce the formation of F-actin rich membrane projections by Cdc42- dependent and independent mechanisms; and 2) that drebrin plays an active role in directing the secretagogue-dependent formation of F-actin-rich filaments on the parietal cell canalicular membrane. Finally, the differential distribution of drebrin in parietal cells along the gland axis suggests that drebrin E2 may be an important marker of parietal cell differentiation and functionality.