The role of extracellular matrix as a determinant of intestinal cell maturation was explored by growing a normal, but immature, rat small intestinal cell line (IEC-6) on basement membrane extract from Engelbreth-Holm-Swarm (EHS) sarcoma cells (ECM). Grown on plastic or glass, these cells are relatively immature and proliferate rapidly. In contrast, cells on ECM attached more rapidly, stopped proliferating, and rapidly organized into multicellular complex structures. Ultrastructurally, cells grown on ECM displayed significantly more mitochondria, rough endoplasmic reticulum, apical microvilli, and complex golgi apparatus, consistent with greater maturity and synthetic activity. By indirect immunofluorescence, sucrase, alkaline phosphatase, and cellular apolipoprotein B were present in cells grown on ECM only. In contrast to cells grown on glass, these cells also demonstrated Na-dependent glucose absorption, a function unique to mature villus cells (7). We conclude that the basement membrane may be a key determinant of intestinal epithelial cell maturation. The development of a mature villuslike intestinal cell in vitro may have wide application for future studies of induction and regulation of intestinal maturation and function.