Ebrotidine is a new H2-receptor antagonist also known for its gastroprotective effect against ethanol-induced mucosal injury. In this study, we investigated the effect of ebrotidine on the activity of the gastric mucosal calcium channels. The channel complex was isolated from the solubilized gastric epithelial cell membranes by affinity chromatography on wheat germ agglutinin. The complex following labeling with [3H] PN200-110 was reconstituted into phosphatidylcholine vesicles which exhibited active 45Ca2+ uptake into intravesicular space and responded in a concentration-dependent manner to calcium channel activator, BAY K8644, as well as to calcium channel antagonist, PN200-100. The 45Ca2+ uptake was inhibited by ebrotidine which caused maximum inhibitory effect of 54.9% at 50 micrograms/ml. The gastric mucosal calcium channels on epidermal growth factor binding (EGF) in the presence of ATP responded by an increase in tyrosine phosphorylation of 55 and 170 kDa proteins, and the vesicles containing the phosphorylated channels displayed a 48% greater 45Ca2+ uptake. This phosphorylation process was inhibited by ebrotidine which also interfered with the binding of EGF to calcium channel protein. The results point towards the importance of EGF in the maintenance of gastric mucosal calcium homeostasis, and suggest that ebrotidine has the ability to protect the cellular integrity from calcium imbalance by modulating the EGF-stimulated gastic mucosal calcium channel phosphorylation.