Previous studies found functional apical and basolateral EGF receptors (EGFR) on primary canine gastric epithelial monolayer cultures that decreased paracellular permeability. This decrease was evident by an increase in transepithelial electrical resistance (TER) and a decrease in the flux of ³H-mannitol (MF). Although apical EGFR are fewer in number, they induce sustained changes in TER and MF, whereas basolateral EGFR cause only transient changes. Studying primary cultures of enzyme-dispersed canine gastric epithelial cells cultured on filters or in Transwell inserts, we now find that treatment with apical, but not basolateral, EGF enhanced tolerance to apical H⁺, evident by a slower decay in TER and an attenuated rise in MF. The ability of apical EGFR to enhance tolerance to apical acid was evident within 10 minutes of treatment, but was particularly marked over prolonged exposure to apical acid. Immunoneutralization of endogenous TGF impaired the ability of monolayers to tolerate apical acidification, as reflected by an accelerated drop in TER and rise in MF; apical EGF treatment restored monolayer resistance to apical acidification. Studying monolayers cultured in Transwell inserts, immunoblockade of basolateral, but not apical, EGFR also impaired the resistance to apical acidification and enhanced MF. We speculate that apical EGFR regulate the barrier to apical acidification via effects on paracellular resistance. Although exogenous basolateral EGF has a lesser apparent effect on the barrier to acid in our model, endogenous ligand active at basolateral EGFR plays an important role in maintaining the barrier to apical acid. Our data implicate an important role for an apical EGFR ligand, which may be EGF or another member of the EGF family.