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Plasma AA in horses fed either an all-hay or a hay and grain diet in a traditional format have not been investigated. Eight horses were divided into 2 groups: a hay group fed only grass hay or a hay and a grain group (HG) fed in a crossover design for two 5-wk periods. After the first period, horses were fasted overnight, followed by feeding with blood sampling every hour for 6 h. A 4-d total fecal and urine collection to evaluate N balance followed. A 10-d washout period separated the 5-wk feeding periods, during which horses switched diets. The second period was also followed by fasting, feeding, blood sampling, and a 4-d collection period. Horses consumed 840 g of CP in the hay group and 865 g of CP in the HG group. Horses in the hay group had a 2.4 ± 2.4 g/d N balance, which was not different from 0 (P = 0.34), whereas horses in the HG group had 5.4 ± 2.4 g/d N balance, which was different from 0 (P = 0.045). Fecal N excretion was greater for the hay group compared with the HG group (hay = 51.1 ± 1.3 g/d and HG = 45.5 ± 1.3 g/d; P = 0.011), and urine N excretion was greater for the HG group compared with the hay group (hay = 79.3 ± 2.8 g/d and HG = 89.2 ± 2.8 g/d; P = 0.026). Plasma AA concentrations were greater in the HG group compared with the hay group for Met (P = 0.001), Lys (P = 0.001), Ile (P = 0.047), Arg (P < 0.001), Gln (P = 0.009), and Orn (P = 0.002). Plasma concentrations were less for the HG group compared with the hay group for Thr (P < 0.001) and Ala (P < 0.001). Plasma concentrations of urea were greater for the HG group compared with the hay group (P < 0.001), whereas 3-methyl-histidine concentrations were greater for the hay group compared with the HG group (P < 0.001). The effect of diet on the excretion of N via feces vs. urine in the hay and HG groups is typical. The early increases in the plasma concentrations of Met, Val, Ile, Leu, Phe, Lys, Arg, and Ala during the postfeeding phase are most likely due to increased foregut digestibility as well as a greater quality AA profile in the grain. The greater concentrations of Thr, Leu, and Val later in the postfeeding phase for the hay group most likely reflects slower digestion because of prolonged consumption time compared with the HG group. Improved N balance observed in the HG group supports the fact that the HG group had more available AA via the AA profile and foregut digestibility of the HG diet. Despite the fact that both groups consumed similar amounts of CP, the AA profile and availability affected N balance.