In many mammals, enterocytes are responsible for the synthesis of L-citrulline and L-arginine from L-glutamine and L-proline. However, there is limited research in the horse to determine de novo synthesis of L-citrulline or L-arginine by the enterocyte, which is the initial step in establishing dietary requirements and comprehending arginine nutrition in horses. We utilized jejunal enterocytes from 20 horses (neonate n = 2, mature n = 16, and geriatric n = 2) to study glutamine and proline metabolism. As a positive control, enterocytes were isolated from 0, 60, and 180-day old pigs (n = 8/age group). Equine or porcine enterocytes were incubated at 37oC for 30 min in oxygenated (95% O2/5% CO2) Krebs bicarbonate buffer (pH 7.4) containing 5 mM D-glucose and either 2 mM L-[U-14C]glutamine or 2 mM L-[U-14C]proline plus 2 mM L-glutamine. Collected 14CO2 was determined by a liquid scintillation counter, whereas amino acids in cells plus medium were analyzed by HPLC. Both equine and porcine enterocytes oxidized glutamine to CO2 but had a limited ability to oxidize proline to CO2, confirming the biochemical viability of the cells in vitro. Enterocytes from neonatal and geriatric horses had lower rates (nmol/106 cells/30 min) of CO2 production (1.10 ± 0.52 and 1.31 ± 0.65, respectively) from glutamine than those (40.9 ± 5.40) from mature horses. Porcine enterocytes synthesized L-citrulline and L-arginine from glutamine and proline (e.g., 4.86 ± 0.26 nmol L-citrulline/mg protein/30 min and 0.85 ± 0.04 nmol L-arginine/mg protein/30 min from glutamine in enterocytes of 60-day-old pigs). In contrast, equine enterocytes did not synthesize L-citrulline and L-arginine from L-glutamine or L-proline. Because L-arginine is an essential substrate for the synthesis of protein, nitric oxide, and creatine, our novel findings on the lack of intestinal synthesis of L-arginine in horses have important implications for their nutrition, metabolism, and health.