Background: The transforming growth factor β (TGF-β) proteins are key regulators of cellular growth and differentiation. Previous studies have shown that TGF-β₁ is a potent growth inhibitor of cultured jejunal epithelial cells. The reported distribution of TGF-β₁ messenger RNA (mRNA) expression along the intestinal villus has been controversial. The purpose of the current study is to determine the loci of TGF-β protein expression in the normal small intestine and colon. Methods: Intestinal localization of TGF-β isoform mRNA and protein was examined by Northern blot analysis and immunohistochemistry using isoform specific reagents. Results: TGF-β₁, TGF-β₂, and TGF-β₃ mRNA were found in homogenates from the intact mouse jejunum and colon. The three isoforms colocalized in these tissues. Expression in the small intestinal epithelium was most prominent in cells located on the villus tip, and no staining was detected in the crypt. Occasional lymphocytes in the lamina propria were immunopositive, and all layers of the muscularis were moderately stained. This pattern was seen in all regions of the small intestine. The surface epithelium of the colon was intensely immunopositive, whereas cells in the glands were only weakly stained. Conclusions: TGF-β molecules may serve overlapping functions in the intestinal tract, and expression in the epithelium may function to arrest growth of cells emerging from the crypt and induce or maintain the terminally differentiated state.