HT-29-A7, a CD4-negative clonal derivative of the human colonic adenocarcinoma cell line HT-29, is particularly sensitive to infection by several isolates of HIV-1 and, correspondingly, expresses high amounts of galactosylceramide (galactocerebroside, GalCer). GalCer is a neutral glycolipid which binds to the HIV-1 envelope glycoprotein gp120 and is present at abundant levels in normal human epithelial cells of the small and large intestine. Treatment of the HT-29-A7 cells with recombinant gamma-interferon (rlFN gamma) induced a dose-dependent inhibition of GalCer expression on the cell surface, as demonstrated by indirect immunofluorescence and by enzymatic labeling of cell surface glycoconjugates with oxidase-tritiated sodium borohydride. The rIFN gamma effect was not associated with any toxicity and was specific for GalCer, since expression of carcinoembryonic antigen did not decrease following treatment. The decrease in GalCer expression was associated with resistance of the cells to HIV-1 infection. In contrast, rIFN gamma did not alter cell surface expression of CD4, the classical HIV receptor, in HT-29-A7 cells that had been transduced with a retroviral vector expressing full-length CD4, and there was no effect on their infection. These results strongly suggest that rIFN gamma blocks HIV-1 infection of HT-29-A7 cells by decreasing GalCer synthesis and expression. This effect on expression of a viral receptor is a novel antiviral property of rIFN gamma which should be exploited for antiviral therapeutics.