The localization of alkaline phosphatase activity in the small intestinal microvilli of the duodenum, jejunum, and ileum in various mammalia, birds, reptilia, amphibia and fishes has been investigated and compared both by light and by electron microscopes. When the incubating medium is intravitally perfused into the small intestine of living animals, deposition of azo-dye is found on the microvilli in the epithelium. Enzyme activity in the duodenal epithelium is strong in all 14 species. No appreciable differences in phosphatase activity are found among the species. In the birds and mammalia except rat, the microvilli of the jejunal and ileal epithelium shows strong alkaline phosphatase activity. In the amphibia and fishes, the jejunal epithelium of the striated border shows weak phosphatase activity. Ileal epithelium of the reptilia, amphibia and fishes shows weak or trace activity. In the glutaraldehyde-fixed material, the phosphatase activity of the duodenum in mammalia is similar in activity to that found in the intravitally perfused animal. But no azo-dye deposit can be seen on the microvilli in the jejunum of amphibia and fishes, nor the ileum of reptilia, amphibia and fishes. Furthermore, the effect of EDTA-inhibition for alkaline phosphatase in the small intestinal surface was also viewed in glutaraldehyde-fixed sections.