The single cell-thick intestinal epithelium forms a crucial barrier between the host and environment, and is modeled in vitro by a monolayer of polarized, highly differentiated T84 epithelial cells impermeable to most macromolecules because of functional intercellular tight junctions. Absence of a permeability defect across the monolayer, either transcellular or paracellular, is indicated by development of a transepithelial electrical resistance of > or = 1000 ohm-cm2, reported to be markedly diminished by exposure to a T lymphocyte cytokine, IFN-gamma. We sought to define this phenomenon in four ways by determining its duration and reversibility; the uniqueness of type II (gamma) IFN as opposed to type I (alpha) IFN; the surface of the polarized columnar epithelium likely involved in responding to IFN-gamma; and whether a specific surface membrane receptor on the epithelial cell participates in the response. Using a special apparatus that allows differential cytokine exposure of monolayer surfaces, our data demonstrate 1) only the monolayer's basolateral surface is IFN-gamma responsive, whereas the apical (microvillous) surface is no; 2) the alteration in electrical resistance of epithelium is prolonged (5 days), even after a single (24 h) exposure to IFN-gamma, but nevertheless is reversible; 3) the effect is likely receptor-ligand mediated, because it can be partially blocked by IFN-gamma receptor-specific monoclonal Ig; 4) an alteration in tight junction function (a paracellular pathway) rather than cell necrosis or a transcellular pathway is responsible for IFN-gamma-induced monolayer dysfunction because permeability to a 44,000-Da macromolecule (horseradish peroxidase) did not increase, and intracytoplasmic T84 cell enzymes were not released into the media; and 5) the biologic phenomenon could not be induced by a species (alpha) of class I IFN, making IFN-gamma reasonably unique in this regard. Given the proximity; activation status, and capacity of T lymphocytes for cytokine production in mucosa, we suggest that IFN-gamma-induced changes in epithelial permeability may be a major cause of altered intestinal barrier function in vivo.