Conditioned culture medium of rat small intestinal epithelial cells suppressed proliferation of spleen lymphocytes stimulated with concanavalin A (approx. 10% of its control [H-3]thymidine incorporation) whereas conditioned phosphate-buffered saline of the epithelial cells did not. On the other hand, conditioned saline of the epithelial cells exposed to a mixture of total 22 amino acids at their concentrations in the culture medium suppressed the proliferation (approx. 45% of its control [H-3]thymidine incorporation). Neither conditioned saline of the epithelial cells exposed to other medium components nor lysates of freshly harvested epithelial cells suppressed the proliferation. Thus, amino acids synergistically stimulated intestinal epithelial cells to produce substances with the ability to suppress lymphocyte proliferation.