After injury and loss of epithelial cells, intestinal barrier function is reestablished by migration of viable epithelial cells from the wound edge (restitution). Myofibroblasts are located close to the basal surface of epithelial cells. This study aimed to investigate the role of human colonic subepithelial myofibroblasts in epithelial restitution. Primary cultures of subepithelial myofibroblasts were established. Monolayers of the epithelial cell lines IEC-6 and T84 were "wounded" in a standard manner to create an in vitro model of restitution. Migration of epithelial cells across the wound edge was assessed following culture in myofibroblast-conditioned medium. Myofibroblast expression of transforming growth factor (TGF)-beta isoforms was examined using RT-PCR, and TGF-beta isoform bioactivity was assessed using Mv 1 Lu bioassay. Myofibroblast-conditioned medium, via a TGF-beta-dependent pathway, significantly enhanced migration of epithelial cells across the wound edge and significantly inhibited cell proliferation in wounded monolayers. Messenger RNA for TGF-beta1, -beta2, and -beta3 was detected in the myofibroblasts, and Mv 1 Lu bioassay showed the presence of predominantly bioactive TGF-beta3. This study shows that human colonic subepithelial myofibroblasts secrete predominantly bioactive TGF-beta3 and enhance restitution in wounded epithelial monolayers via a TGF-beta-dependent pathway.