The amino acid requirements of growing horses are currently unknown, and studies suggest that threonine is a limiting amino acid in common horse diets. Thus, the objective of this study was to determine the threonine requirement of growing horses fed two different forage to concentrate ratios using the indicator amino acid oxidation method. The study consisted of a high concentrate phase (HC; 60% concentrate and 40% forage) and a high forage phase (HF; 25% concentrate and 75% forage). Within each phase, 6 female yearling Thoroughbred horses were randomly assigned each of 6 dietary treatments in a 6 x 6 Latin square design. All 6 treatments were identical, apart from varying equimolar ratios of threonine to glutamate. After 6 days of adaptation, blood samples were collected before and after the morning meal for plasma urea nitrogen (PUN) and amino acid analysis. On day 7, horses underwent the IAAO protocol, during which regular breath and blood samples were collected. Phenylalanine flux, oxidation, non-oxidative disposal, and release from body protein, as well as total carbon dioxide production were calculated using plateau enrichment of samples. There was a significant linear effect of threonine intake on plasma threonine concentrations, and PUN had a significant linear response during the HC phase. There was no significant effect of treatment on phenylalanine oxidation during either phase (P ≥ 0.05). It is unlikely that threonine was limiting in the experimental diets.