Mucin-type glycoproteins are the major structural proteins in gastric mucus. Stomach mucin proteins include MUC5AC, synthesized by surface foveolar or pit cells, and MUC6, synthesized by neck and gland cells. The aim of this study was to determine the spatial distribution of these mucin proteins within the extracellular mucous coat. Double-labeling immunoflourescence/confocal microscopy was used in histologically normal surgical resection specimens. Intralumenal mucin within antral glands consisted entirely of MUC6 protein. Intralumenal mucin within the gland isthmus region consisted of an irregular mixture of MUC5AC and MUC6. The mucous layer on the gastric surface consisted primarily of MUC5AC extending in layered sheets with MUC6 protein layered in between. The laminated appearance of the surface mucus was present in both H. pylori-infected and noninfected specimens. These data indicate that MUC5AC and MUC6 proteins remain segregated within the mucous gel in a laminated linear arrangement. The physical stratification of mucin proteins may confer increased strength to the mucous layer or represent independent and redundant protection.