Intestinal smooth muscle cells play a major role in the stricture formation that complicates chronic intestinal inflammation, by proliferating and producing collagen. Transforming growth factor β₁ has been identified as an important inflammatory mediator in the fibrotic response of human tissue to inflammation. To determine whether this mediator might be involved in intestinal fibrosis, the effect of transforming growth factor β₁ on collagen production and proliferation by human intestinal smooth muscle cells was studied in vitro. Cells in the second passage were grown to subconfluence in medium containing 10% Nu-Serum (Collaborative Research Inc., Bedford, MA), after which the concentration of Nu-Serum was decreased. Forty-eight hours later, transforming growth factor β₁ was added to the culture medium to achieve concentrations of 1–500 pmol/L. After 24 hours exposure to the transforming growth factor β₁, cellular collagen synthesis was determined by the uptake of [³H]proline into collagenase-sensitive protein. Transforming growth factor β₁ caused a 100% increase in collagen production and a 40% increase in noncollagen protein production per cell, reflecting an increase in relative collagen synthesis of 58%. This effect was maximal at a concentration of 10 pmol/L. Epidermal growth factor, by comparison, had no significant effect on relative collagen synthesis. Transforming growth factor β₁ caused a significant increase in the uptake of methylaminoisobutyric acid, a nonmetabolized amino acid analog, into the cells at 10 pmol/L. However, this effect was small (20% increase) compared with the effect on the uptake of proline into collagen (100% increase) at this concentration. When cell proliferation was examined by the uptake of [³H]thymidine, transforming growth factor β₁ had no effect, whereas epidermal growth factor (1000 pmol/L) caused a 94% increase. Transforming growth factor β₁ selectively augments collagen production by human intestinal smooth muscle cells in vitro. This effect is potent and is not related to effects on either cell proliferation or amino acid uptake. These data suggest that transforming growth factor β₁ has an important role as an inflammatory mediator in the pathogenesis of intestinal fibrosis.