Maintenance of the integrity of the single-cell-thick intestinal epithelium as an in vivo barrier between environmental Ags and mucosal immunocytes is pivotal for health. The T cell cytokine IFN-gamma consistently disrupts this epithelial barrier in vitro, but the substances in mucosa that may be responsible for sustaining or enhancing barrier function have not been clearly identified. Therefore, we characterized the effect on the epithelial barrier of TGF-beta 1 and three prominent neuropeptides (VIP, substance P, somatostatin) by using a model system in which barrier function of a mature polar human colonic epithelial (T84) cell monolayer is reflected in 1) the electrical potential difference across the apical to basolateral surface of each cell, 2) the transmonolayer permeability to macromolecules such as horseradish peroxidase, and 3) lactate dehydrogenase release into the medium indicating epithelial cell cytolysis. Whereas T84 monolayers exposed to TGF-beta 1 alone demonstrated a modest increase in electrical resistance and barrier integrity, TGF-beta 1 showed a striking ability to reduce the capacity of IFN-gamma to disrupt epithelial barrier function. Characterization studies demonstrated that this TGF-beta 1 effect was prolonged (e.g., days) after a single exposure, progressive over the dose range 0.1 to 2.5 ng/ml, reversible with increased concentrations of IFN-gamma, and more pronounced when TGF-beta 1 exposure was to basolateral rather than to apical epithelial membranes. Macromolecular (horseradish peroxidase) penetration of epithelium was not simultaneously altered by TGF-beta 1 and epithelial cellular injury was minimal as gauged by lactate dehydrogenase release. Additional studies using a human pathogen demonstrated that TGF-beta 1 delayed and decreased the barrier disruption caused by exposure to Cryptosporidium parvum. TGF-beta 1 may be the first of a new class of cytokines that maintains and/or enhances barrier function of human enterocytes, in part by countering the effect of a T cell cytokine.