Hydrophilic gel permeation chromatography of 14-36 wk human amniotic fluid on Fractogel columns divides the total alkaline phosphatase (AP) activity in a higher and a lower mol wt zones. Differential inhibition testing, isoelectric focusing, cellulose acetate, agarose and polyacrylamide gel electrophoreses before and after neuraminidase treatment show the higher mol wt zone to be homogeneous and to be made of the higher mol wt foetal intestinal isoenzyme form whereas the lower mol wt zone represents an unresolved mixture of hepatic, placental and lower mol wt foetal intestinal isoenzymes. In the early stages of pregnancy, the activity associated with the higher mol wt zone outweighs by far that of the lower mol wt zone; however from the 24 th week one notes a steady increase in the relative magnitude of this second zone until at the end of the gestation period both zones assume near equal importance albeit within a lower total AP activity. Satisfactory quantitation of the higher mol wt foetal intestinal isoenzyme form in one ml amniotic fluid can be attained after a 3-h chromatography run using p-nitrophenylphosphate as substrate.