Efficient protein synthesis plays an important role in the physiology and pathophysiology of the human gastrointestinal tract. Because of methodological restrictions, no studies on ileal protein synthesis in situ are available in humans. We used advanced mass spectrometry techniques (capillary gas chromatography/combustion isotope ratio mass spectrometry) to determine directly the incorporation rate of [1-¹³C]leucine into ileal mucosal protein in control subjects and postoperative patients. All subjects had an ileostomy, which allowed easy access to the ileal mucosa. To examine changes in ileal protein synthesis during prolonged isotope infusion (0.16 µmol · kg¹ · min¹, 9.6 µmol/kg prime), studies were performed over a 10-h period. Mucosal biopsies were performed after 3, 6, and 10 h of infusion. Protein synthesis was calculated separately between hour 3 and hour 6 (period 1) and hour 6 and hour 10 (period 2). Control subjects demonstrated an ileal protein fractional synthetic rate of 0.62 ± 0.06%/h in period 1 and of 0.52 ± 0.08%/h in period 2 (not significant). In postsurgical subjects, ileal protein synthesis was significantly higher (1.11 ± 0.14%/h in period 1, P < 0.01 vs. controls in period 1) but declined markedly in period 2 (0.39 ± 0.13, P < 0.01 vs. period 1 after surgery). The rate of protein synthesis in the small bowel of control subjects is, thus far, among the lowest measured in mammals and reflects the comparably slow turnover of human ileal mucosa. Postoperative disturbances of gut integrity lead to an accelerated anabolic response. During prolonged isotope infusion, stimulated protein synthesis declines because of diurnal variations or is erroneously reduced by tracer loss due to an accelerated cell turnover.