Synthesis of fibronectin in an epithelial cell line (IEC-6) established from rat small intestine was demonstrated by using immunofluorescence, radioimmunoassay, and collagen-binding. Internally labeled radioactive fibronectin isolated from the IEC-6 cells gave a single main band in sodium dodecyl sulfate/polyacrylamide gel electrophoresis under reducing conditions. Fibronectin isolated from rat plasma gave two closely spaced bands. The slower one had the same mobility as the epithelial cell fibronectin. The distribution of fibronectin in IEC-6 cells as detected by immunofluorescence was different from that described for fibroblasts and other cell types; fibronectin was present exclusively in regions of cell-to-cell contact. No fluorescence was detected on the surface membrane facing the culture medium or underneath the cells. This suggests that fibronectin may not be involved in the adhesion of the epithelial cells to the growth surface but could mediate cell-to-cell contacts. In microscopic sections of the small intestine, immunofluorescent staining with antifibronectin serum was strong in the basement membrane underlying the epithelial cells in the crypts. The in vitro synthesis of fibronectin by the crypt cells and its abundant presence in the basement membrane underlying the same cells in vivo suggests that fibronectin is a structural component of the basement membrane, and that it may be, at least in part, synthesized and deposited by the intestinal epithelium.