The cellular localization of tryptophan hydroxylase (TPH) in the gastrointestinal tract of mice was immunohistochemically investigated in comparison with that of serotonin. The anti-TPH antiserum used selectively stained serotonin-containing enterochromaffin (EC) cells in mucosal epithelium. The immunoreactivity for TPH was also found in nerve fibers of the submucosal and muscle layer, but this was presumed to result from a cross-reaction of the anti-TPH antiserum with tyrosin hydroxylase (TH) contained in adrenergic neurons. All TPH-immunoreactive cells throughout the gastrointestinal tract were identical to EC cells, although the intensity of the immunoreactivity conspicuously varied from cell to cell. In the ileum and colon, the immunoreactivity was only weakly-positive or even undetectable in part of the EC cells. Possible reasons for this heterogeneous immunostaining were described. Immunohistochemistry at the electron microscopic level demonstrated that the TPH immunoreactivity was localized diffusely in the cytoplasmic matrix and also in some of the secretory granules. EC cells were few in the gastric body, the major population of serotonin-containing cells there being mast cells distributed in the lamina propria and tela submucosa. Noteworthily, these mast cells were immunonegative for TPH, and possible reasons for this unexpected finding are discussed.