Human intrinsic factor (IF) is found on the surface of parietal cells at the light microscopic level by immunofluorescence, but there is no direct evidence that IF is synthesized in and secreted from this cell. In order to provide this evidence, IF was localized at the ultrastructural level in human stomach, using an immunochemically, monospecific antibody against purified human IF. We defined optimal immunocytochemical conditions and identified the subcellular location of IF in fundic biopsies from 4 fasting subjects. Intrinsic factor was found on the perinuclear membrane, on rough endoplasmic reticulum, Golgi apparatus, tubulovesicles, and membranes of the multivesicular body in parietal cells. Intrinsic factor was absent from nuclei, mitochondria, basolateral surface membranes, blunt microvilli, and the cytosol. Thus, we have defined the parietal cell as the site of IF synthesis. The observed distribution of intracellular IF suggested that an interrelationship existed between the stained membranous structures, and that IF secretion may depend upon membrane translocation. Further immunocytochemical and physiologic studies should increase our understanding of the cellular physiology of IF secretion as well as the parietal cell.