Abstract Biological characteristics of alkaline phosphatase (ALP) from rat intestine were compared among four various fractions (brush border, cytosol, luminal and lymph) to clarify the source and route of ALP appeared in intestinal lymphatics. ALP of each fraction had the same K m values and the same heat stability and all were of intestinal type. However, lymph and cytosol ALP showed similar affinity to three kinds of lectins and were distinct from membrane and luminal ALP. On polyacrylamide gel electrophoresis (PAGE), there seems to be two types of charge isomers of intestinal ALP, namely brush border type and lymph type. On sodium dodecyl sulfate (SDS)-PAGE, the molecular weight of principal band for each form was: 130,000 for brush border type, and 160,000 for lymph type. Fat administration significantly increased the ALP activity in intestinal lumen and intestinal lymph. However, it did not change biological characteristics of ALP including binding ability to lectins and electrophoretic mobility in any of these four fractions. These results suggest that lymphatic ALP may be transported from cytosol of intestinal cells and that fat administration seems to induce quantitative change of intestinal ALP, but not to change its physiological route of transportation into intestinal lymph.