Primary cultures of human brain microvessel endothelial cells were used to study the effects of human recombinant interferon-gamma (IFN-gamma) on cerebral endothelium in vitro. Incubation of monolayers with various concentrations of IFN-gamma (10 to 200 U/ml) for 12 to 96 hours induced surface expression of class II major histocompatibility complex (Ia) antigen in a time- and concentration-dependent manner. In immunogold-stained cultures, labeling was observed as early as 12 hours, was maximal after 48 hours, and persisted at plateau levels in the continuous presence of the cytokine. Expression was blocked by coincubation with anti-IFN-gamma antibody and was reversed 4 days following removal of IFN-gamma from the culture media. Endothelial cells treated with IFN-gamma for 3 to 4 days became spindle-shaped, extensively overlapped, and frequently formed cellular whorls. These changes did not occur in the presence of anti-IFN-gamma antibody and reversed upon removal of IFN-gamma from the media. The morphological alterations were associated with increased permeability of confluent monolayers to macromolecules as compared with untreated cultures. The results of these studies indicate that human brain microvessel endothelial cells respond to in vitro cytokine stimulation by undergoing profound morphological, functional, and permeability changes. We conclude that cerebral endothelium may play an important role in the initiation and regulation of lymphocyte traffic across the blood-brain barrier in inflammatory disorders of the human central nervous system.