The formation of intestinal villi (organogenesis phase) may be studied in organ culture with a completely synthetic medium in 15-day fetal mouse duodenal explants. However, in these explants absorptive cells remained poorly differentiated with all the hormones studied except with epidermal growth factor. In order to elucidate the role of hormones and other factors on the maturation of absorptive cells (maturation phase) in the fetal rodent in organ culture, we have taken the explants after the organogenesis phase. We have studied different culture conditions and have found that 17-day mouse duodenal explants can be cultured during 48 hours with Leibovitz L-15 medium in a 95% O2-5% CO2 atmosphere provided that the explants are relatively large (5 × 2 mm). With this method, dexamethasone (Dx) has been shown to have a direct effect on the maturation of the fetal duodenal mucosa. The addition of Dx (300 ng/ml) to the completely synthetic medium (1) improves the morphology of the explants, (2) induces a significant increase in maltase activity in the tissues, and (3) reduces significiantly the labeling index of the duodenal explants after 48 hours of culture. Direct action of Dx on the duodenal mucosa is shown for the first time in organ culture using a completely synthetic medium. This method will permit us to study the effects of other intrinsic and extrinsic factors on the regulation of enzymatic maturation in fetal small intestine.