Salmonella cytotoxin present in cell lysates inhibited protein synthesis in both Vero cells and isolated rabbit intestinal epithelial cells. A time-course experiment with Vero cells revealed progressive inhibition of protein synthesis beginning as early as 1 h after exposure to the Salmonella cell lysate. Significant loss of membrane integrity of Vero cells (measured by release of [3H]uridine) did not appear before 24 h. To demonstrate inhibited protein synthesis in intestinal tissue during experimental salmonellosis, we isolated rabbit epithelial cells from Salmonella-infected and control intestinal loops. Measurement of [3H]leucine incorporation showed a similar decrease in protein synthesis in epithelial cells from the Salmonella-infected intestinal loop as that from an adjacent control loop. A similar pattern of protein synthesis inhibition was observed when isolated epithelial cells from normal rabbit intestine were exposed to the Salmonella cell lysate. The inhibited protein synthesis in the intestinal cells provides a molecular basis for the cellular damage caused by Salmonella cytotoxin during experimental salmonellosis.