Previous studies have shown that human leukocyte interferon (IFN), IFN-gamma, and recombinant (r) IFN-alpha-A protect cultured human cells against the invasiveness of salmonellae, shigellae, and enteroinvasive Escherichia coli. To differentiate between effect on endocytosis and adhesiveness, HEp-2 cells were pretreated with cytochalasin B to block endocytosis, and thereafter treated with IFN. Although a significant reduction of adhesion of bacteria to HEp-2 cells was observed, this reduction was far too small to explain the total reduction of bacterial invasiveness. Thus, the anti-invasive effect of IFN on HEp-2 cells is due mostly to inhibition of endocytosis. The IFN effect was not expressed at 4 degrees C, and the ribosomal 60S subunit inactivators, cycloheximide, abrin, and shigella toxin, completely blocked the expression of the IFN-induced anti-invasive state of the cell. Thus, the IFN effect is dependent on continuous ribosomal activity. Our results indicate that the major effect of IFN on bacterial invasiveness is on endocytosis, and that the action is mediated through the effect of IFN-induced protein synthesis.