To assess the effect of glutamine availability on rates of protein synthesis in human enterocytes, Caco-2 cells were grown until differentiation and then submitted to glutamine deprivation produced by exposure to glutamine-free medium or methionine sulfoximine [L-S-[3-amino-3-carboxypropyl]-S-methylsulfoximine (MSO)], a glutamine synthetase inhibitor. Cells were then incubated with ²H₃-labeled leucine with or without glutamine, and the fractional synthesis rate (FSR) of total cell protein was determined from ²H₃-labeled enrichments in protein-bound and intracellular free leucine measured by gas chromatography-mass spectrometry. Both protein FSR (28 ± 1.5%/day) and intracellular glutamine concentration (6.1 ± 0.6 µmol/g protein) remained unaltered when cells were grown in glutamine-free medium. In contrast, MSO treatment resulted in a dramatic reduction in protein synthesis (4.6 ± 0.6 vs. 20.2 ± 0.8%/day, P < 0.01). Supplementation with 0.5-2 mM glutamine for 4 h after MSO incubation, but not with glycine nor glutamate, restored protein FSR to control values (24 ± 1%/day). These results demonstrate that in Caco-2 cells, 1) de novo glutamine synthesis is highly active, since it can maintain intracellular glutamine pool during glutamine deprivation, 2) inhibition of glutamine synthesis is associated with reduced protein synthesis, and 3) when glutamine synthesis is depressed, exogenous glutamine restores normal intestinal FSR. Due to the limitations intrinsic to the use of a cell line as an experimental model, the physiological relevance of these findings for the human intestine in vivo remains to be determined.