Leucylnaphthylamidase, an enzyme bound to the outer surface of the microvilli of the small intestine, has been studied by quantitative and topographic means in the fetal, infant, and adult mouse. Specific activity in the mature jejunum is about 3 times that in the duodenum, and is also somewhat higher than in the ileum. In the posterior 4 5 of the intestine specific activity is consistently higher in females than in males, and total activity is very significantly greater in the former. LNA activity rises rapidly during the last 4 days of prenatal life, then falls slightly to a minimum at about 10 days after birth. In the middle and distal thirds of the intestine, activity rises steadily from 14 to 22 days, and then falls to the adult levels. In the adolescent duodenum, activity undergoes only a slight increase that is not sustained. At 22 days, activity per gram body weight is 20% greater than in mature females. Histochemical observations reveal that up to 12 days activity is confined to the villi bases. By 16 days it spreads to the tips in the jejunum, but retains its basal localization in the duodenum. Cortisone acetate administered at 4 days elicits a nonsignificant increase in LNA activity in duodenum and jejunum at 6 days; if administered at 9 days, the hormone elicits a greater increase in both parts of the intestine, the differences being highly significant. In the duodenum the increase is however transient. Actinomycin D administered at 10 days raises jejunal LNA activity to 149% of the control value at 13 days; this treatment has no effect on duodenal LNA although it more than doubles duodenal alkaline phosphatase activity. Cortisone acetate and actinomycin D administered in combination have an additive effect in jejunum, but no effect in duodenum. Cycloheximide appears to work synergistically with cortisone in the jejunum. In the duodenum this combination of agents also significantly raises LNA activity in the 10–13-day interval, presumably by stabilizing the transient increase induced by the hormone. Histochemical observations indicate that the additivity of the effects of cortisone and actinomycin D results from their ability to act at different sites. After actinomycin D administration LNA stainability is strongly enhanced but remains limited to the basal parts of the villi. Addition of cortisone permits the increased activity to spread to the villi tips. Comparison of the present results with those previously obtained in similar studies on alkaline phosphatase supports the hypothesis that the differentiation of diverse enzymes may be controlled by common epigenetic influences, which in this case may act primarily on the character of the microvillous membrane.