Monospecific antibody to purified alkaline phosphatase has been used to localize alkaline phosphatase in the rat small intestine at the light microscopic level. Pieces of duodenum, jejunum, and ileum were removed from 2-, 9-, 12-, 18-, 21-, 26-day-old and adult Wistar rats. They were fixed in Bouin's fluid and examined for the presence of alkaline phosphatase by the immunoperoxidase method. Slides were graded blindly for the intensity of staining. The localization of alkaline phosphatase by the immunoperoxidase method extends previous histochemical observations in several ways. First, diffuse cytoplasmic staining is present particularly in the opical portion of the villus and crypt epithelium. Second, staining for alkaline phosphatase is present on the brush border and in the apical portion of the deep crypt cells throughout the duodenum, jejunum, and ileum at the various ages tested. Third, in the adult rat distal ileum there is more staining on the brush border of the deep crypt epithelial cells than on the villus absorptive cells. These observations are consistent with the presence of a non-brush border alkaline phosphatase in all intestinal cells and with an enzymatically inactive form of alkaline phosphatase in the deep crypt epithelium.