Proximal and distal parts of the small intestine of 8-day-old suckling mice can best be maintained for 48 h in a serum-free organ culture system, Leibovitz L-15, at room air and room temperature. As determined by light and electron microscopy, the villous architecture was preserved as well as the classical ultrastructure of the enterocytes. Incorporation of 3H-thymidine and 3H-leucine continued during the culture period, reflecting a sustained synthesis of DNA and proteins for at least 48 h. The hydrolytic activities of the brushborder membrane, namely of lactase (L), trehalase (T), glucoamylase (GA) and alkaline phosphatase (AlPase) were measured in the explants as well as the culture medium. The overall enzymatic activities were increased as compared to the controls. In the tissue, L, GA and T activities remained stable or even increased during culture while in the medium an accumulation of enzymatic activities was noted especially for GA an AlPase. These results show that the morphological as well as the functional integrity of the mucosa is preserved for at least 48 h when small intestine of suckling mice is cultured in a serum-free medium.