The regulatory effect of epidermal growth factor (EGF) on the developmental pattern of brush border hydrolases was studied in the proximal jejunum and colon of the newborn rat. In the proximal colon, daily administration of EGF for 1, 3, or 5 days postpartum inhibited the postnatal increase in lactase, maltase, and aminopeptidase specific activities. In contrast, in the jejunum EGF did not influence lactase activity, inconsistently increased maltase activity, and partly prevented the early postnatal decrease in aminopeptidase activity. In the proximal colon, EGF showed additive effects with T4 and hydrocortisone on the inhibition of lactase activity. In the jejunum, EGF potentiated the effect of hydrocortisone and T4 on the expression of sucrase activity and had only a slight effect when injected alone. The incorporation rate of [3H]thymidine in the proximal colon and jejunum was not different in control and treated rats, indicating the absence of an effect of EGF on DNA synthesis. These results show that EGF may play an important physiological role in the enzymatic differentiation of the developing intestine during early postnatal development. Alone or acting with T4 or glucocorticoids, EGF may induce the decline of digestive hydrolases in the proximal colon. In the small intestine EGF may play a major role in the triggering of sucrase expression.