The influence of glutamine on protein synthesis in small-bowel enterocytes was tested. Enterocytes were isolated from different levels of the villi of rat jejunum and were incubated in the presence of different glutamine concentrations, up to 3.4 mmol/L. Protein synthesis was determined by measuring incorporation of 3H-phenylalanine into trichloroacetic acid-precipitated proteins. Glutamine, but no other amino acids, stimulated protein synthesis in enterocytes from all levels of the villi. A maximal effect was noted at a glutamine concentration of 0.67 mmol/L, which is the normal plasma concentration. The amino acid stimulated the synthesis of both secreted and nonsecreted proteins. The stimulatory effect of glutamine on protein synthesis was blocked by the glutaminase inhibitor 6-diazo-5-oxo-L-norleucine and was duplicated by equimolar concentrations of acetoacetate or 3-hydroxybutyrate. The results suggest that glutamine stimulates protein synthesis in small-bowel enterocytes and that this effect of glutamine is related to provision of energy. The findings are important because they suggest that increased protein synthesis may be one of the mechanisms by which glutamine exerts its protective effect on gut mucosa during critical illness.