Colonic and intestinal alkaline phosphatase were studied histoenzymatically and biochemically in normal mucosae from eight hemicolectomy specimens. Histochemically, the most intense reaction was found in the small intestine, followed by vascular endothelium and colonic epithelium, respectively. The reaction was stronger in frozen sections than in acetone-fixed specimens, weakest in formalin-fixed tissues, and best when cold fixatives and low melting point paraffin were used. In the small intestine, the reaction was most marked on the villus tips. In the colon, the crypt necks showed the strongest reaction although good staining of luminal epithelium was found with more prolonged incubation. The addition of phenylalanine to the substrate medium resulted in selective inhibition of epithelial staining, whereas levamisole selectively inhibited the vascular reaction. On cellulose acetate electrophoresis, colonic and intestinal alkaline phosphatase showed slightly different rates of migration suggesting the presence of different isoenzymes. Quantitative analysis indicated that the intestinal enzyme expressed in U/g wet tissue was 7.7 times the colonic enzyme in amount. It is concluded that despite its relatively low biochemical activity, alkaline phosphatase can be demonstrated histochemically in colonic epithelium if tissues are processed at low temperatures and if the appropriate fixatives and adequate times are used.