Glutamine has been shown to be an important metabolic substrate of enterocytes in many animals, including cats, dogs, hamsters, human beings, monkeys, rabbits, rats, and sheep. To determine whether glutamine is important in the metabolism of cells of the equine gastrointestinal tract, we examined transintestinal differences in glutamine concentrations in the arterial and venous circulation, and measured activity of the major glutamine catabolizing enzyme, glutaminase. Arteriovenous differences provide an index of the amount of a given substrate removed by the tissue across which the measurements are made, and commonly are expressed as a percentage of substrate removed, or percent extraction. Arteriovenous differences for glutamine were determined in 7 anesthetized adult horses (weight, 450 to 500 kg) before and after an IV glutamine infusion. The mean baseline arterial glutamine concentration (+/- SEM) was 572 +/- 24 microM; this concentration quadrupled (to 2,167 +/- 135 microM, P < 0.01) 1 minute after IV bolus infusion of a 17.5-g glutamine load. Baseline extraction by the portal-drained viscera was 7.5 +/- 1.5%; this value increased to 18 +/- 2% at 1 minute (P < 0.01) and had returned to baseline values 60 minutes later. Arteriovenous differences were greatest across the jejunum (11.8 +/- 1.8% in the baseline period vs 33.1 +/- 3.1% at 1 minute, P < 0.001), with smaller differences across the colon, suggesting that the jejunum was the more avid utilizer of glutamine. Glutaminase activity was 4.38 +/- 0.16 and 4.00 +/- 0.60 micromol/mg of protein/h under standard conditions in jejunal and ileal mucosa, respectively. Kinetic studies of jejunal glutaminase revealed the enzyme to have a Km of 3.81 +/- 0.35 mM and a Vmax of 8.08 +/- 0.54 micromol/mg of protein/h, suggesting that the small intestine of horses has a high capacity to extract and metabolize circulating glutamine.