OBJECTIVE: To determine the contribution of liver and viscera to splanchnic cortisol production in humans. RESEARCH DESIGN AND METHODS: D4 cortisol was infused intravenously; arterial, portal venous, and hepatic venous blood was sampled; and liver and visceral fat were biopsied in subjects undergoing bariatric surgery. RESULTS: Ratios of arterial and portal vein D4 cortisol/cortisol(total) (0.06 +/- 0.01 vs. 0.06 +/- 0.01) and D4 cortisol/D3 cortisol (1.80 +/- 0.14 vs. 1.84 +/- 0.14) did not differ, indicating that no visceral cortisol production or conversion of D4 cortisol to D3 cortisol via 11beta-hydroxysteroid dehydrogenase type 1 (11beta-HSD-1) occurred. Conversely, ratios of both D4 cortisol to cortisol(total) (0.05 +/- 0.01; P < 0.05) and D4 cortisol to D3 cortisol (1.33 +/- 0.11; P < 0.001) were lower in the hepatic vein than in the portal vein, indicating production of both cortisol and D3 cortisol by the liver. The viscera did not produce either cortisol (-8.1 +/- 2.6 microg/min) or D3 cortisol (-0.2 +/- 0.1 microg/min). In contrast, the liver produced both cortisol (22.7 +/- 3.90 microg/min) and D3 cortisol (1.9 +/- 0.4 microg/min) and accounted for all splanchnic cortisol and D3 cortisol production. Additionally, 11beta-HSD-1 mRNA was approximately ninefold higher (P < 0.01) in liver than in visceral fat. Although 11beta-HSD-2 gene expression was very low in visceral fat, the viscera released cortisone (P < 0.001) and D3 cortisone (P < 0.01) into the portal vein. CONCLUSIONS: The liver accounts for all splanchnic cortisol production in obese nondiabetic humans. In contrast, the viscera releases cortisone into the portal vein, thereby providing substrate for intrahepatic cortisol production.