Actinomycin D elevates the alkaline phosphatase activity of the duodenum of the mouse. The effect is relatively slight, but positive, during the first postnatal week, but thereafter becomes marked, not only in the period before the normal maximal level of phosphatase activity is reached at 20 days, but also afterwards, as the activity declines to the adult level. The effect is not manifested in mice at the age of one month, but reappears in adults. Doses of actinomycin that enhance duodenal phosphatase activity depress the activity of the same enzyme in the kidney.Puromycin also increases duodenal phosphatase activity, the effect again being quite small in the first week. At all stages from nine days to adult, however, puromycin strongly elevates activity. Cycloheximide is also effective in raising duodenal phosphatase activity in young mice.Glucocorticoids are known to control the normal developmental increase of phosphatase, but the effect of actinomycin D can be exerted in adrenalectomized mice. The effects of both actinomycin and puromycin are additive with those of hydrocortisone acetate, and both raise phosphatase activity after 15 days, when exogenous corticoids no longer exert any influence.Puromycin stops mitosis in the duodenal epithelium in doses that elevate phosphatase. Hence, some alteration of the enzyme must occur in the striated border of cells that have emerged from the crypts and are in place on the surfaces of the villi. Since the phosphatase-stimulating agents are all inhibitors of protein synthesis, the results are interpreted to mean that duodenal phosphatase normally tends to undergo an activity-enhancing conversion that is restrained by a labile protein.