IQGAPs are thought to regulate a range of actin cytoskeleton-based activities through interactions with small GTPase family members, Cdc42 and Rac. Recently, Cdc42 was implicated in the regulation of gastric parietal cell HCl secretion, and IQGAP2 was immunolocalized along with Cdc42 to F-actin rich intracellular canalicular membranes of isolated gastric parietal cells in primary culture. In this study, we sought to define the distribution and localization of IQGAPs 1 and 2 in the major oxyntic (acid-secreting) gastric mucosal cell types and to determine if secretory agonists modulate these proteins. Differential staining protocols were used to identify different cell populations in semi-intact glands isolated from rabbit gastric mucosae and to characterize these same cells after dispersion and fractionation on isopycnic density gradients. Parietal, chief, surface/pit, and mucous neck cells could be distinguished in glands and in dispersed cell fractions with simultaneous staining for F-actin, H⁺, K⁺-ATPase, and GSII lectin binding sites. There was a pronounced increase in intracellular F-actin staining in dispersed chief cells, apparently resulting from internalization of F-actin rich apical membranes that normally abut the gland lumen. This suggested other membrane-associated proteins might also be redistributed by disruption of cell-cell contacts. Thus, Western blot analyses were used to quantitate relative concentrations of IQGAPs in defined mucosal cell fractions and gastric glands were used for in situ localizations. These approaches detected uniform levels of IQGAP2 expression in oxyntic mucosal cells with predominant targeting to regions of cell-cell contact and nuclei of all cell types in glands. IQGAP2 was not detected in parietal cell intracellular canaliculi. In contrast, IQGAP1 expression was variable and targeted predominantly to the cortex of chief and mucous neck cells. Parietal cells expressed little or no IQGAP1 as compared to other mucosal cell types. Phosphoprotein affinity chromatography, isoelectric focusing and phosphorylation site analyses indicated that both IQGAP1 and IQGAP2 are phosphoproteins with the potential to be regulated by the [Ca²⁺]i/PKC and cAMP signaling pathways, respectively. Stimulation of glands with the cholinergic agonist, carbachol, which elevates [Ca²⁺]i and activates protein kinase C (PKC), induced the apparent translocation of IQGAP1, but not IQGAP2, to the apical poles of chief (zymogen) and mucous neck cells. This response was mimicked by the protein kinase C activator, phorbol myristate acetate (PMA), but not by the calcium ionophore, ionomycin, or by elevation of [cAMP]i with the adenylyl cyclase, activator, forskolin. Collectively, our observations support a novel, PKC-dependent role for IQGAP1 in regulated exocytosis and suggest that IQGAP2 may play a more general role in regulating cell-cell interactions and possibly migration within the gastric mucosa.