We performed ultrastructural immunogold localization of osteopontin in the mucosa of human stomach. This adhesive glycoprotein was present in mucous and chief cells of the epithelial layer and in macrophages in the lamina propria. Parietal and endocrine cells of the epithelial layer and mast cells and plasma cells in the lamina propria did not contain osteopontin, serving as internal negative controls. Subcellular localizations of osteopontin included secretory granules and synthetic organelles in mucous and chief cells and phagolysosomes in macrophages. Extracellular concentrations of osteopontin were present in the glycocalyx and in an electron-lucent band between epithelial surface cells and the gastric lumen. Paracellular edema between the epithelium of the same cells was devoid of osteopontin. Immunogold localization of pepsinogen II was done to identify cells with mixed granule populations and contents of multicompartmental secretory granules. These studies revealed mucous cell granules and chief cell granules, each containing compartmentalized storage products, which included osteopontin and mucigen in mucous cells and osteopontin and pepsinogen II in chief cells. Cytochemical controls for the immunogold localizations were negative. The subcellular distribution of osteopontin in human gastric mucosa suggests possible roles for this glycoprotein in barrier function, host defense, and/or secretion.