The neonatal gastrointestinal tract extracts the majority of dietary threonine on the first pass to maintain synthesis of threonine-rich mucins in mucus. As dietary threonine becomes limiting, this extraction must limit protein synthesis in extraintestinal tissues at the expense of maintaining protein synthesis in mucin-producing tissues. The objective was to determine the dietary threonine concentration at which protein synthesis is reduced in various tissues. Twenty Yucatan miniature piglets (10 females; mean ± SD age, 15 ± 1 d; mean ± SD weight, 3.14 ± 0.30 kg) were fed 20 test diets with different threonine concentrations, from 0.5 to 6.0 g/100 g total amino acids (AAs; i.e., 20-220% of requirement), and various tissues were analyzed for protein synthesis by administering a flooding dose of [3H]phenylalanine. The whole-body requirement was determined by [1-14C]phenylalanine oxidation and plasma threonine concentrations. Breakpoint analysis indicated a whole-body requirement of 2.8-3.0 g threonine/100 g total AAs. For all of the non-mucin-producing tissues as well as lung and colon, breakpoint analyses indicated decreasing protein synthesis rates below the following concentrations (expressed in g threonine/100 g total AAs; mean ± SE): gastrocnemius muscle, 1.76 ± 0.23; longissimus dorsi muscle, 2.99 ± 0.50; liver, 2.45 ± 0.60; kidney, 3.81 ± 0.97; lung, 1.95 ± 0.14; and colon, 1.36 ± 0.29. Protein synthesis in the other mucin-producing tissues (i.e., stomach, proximal jejunum, midjejunum, and ileum) did not change with decreasing threonine concentrations, but mucin synthesis in the ileum and colon decreased over threonine concentrations <4.54 ± 1.50 and <3.20 ± 4.70 g/100 g total AAs, respectively. The results of this study illustrate that dietary threonine is preferentially used for protein synthesis in gastrointestinal tissues in piglets. If dietary threonine intake is deficient, then muscle growth and the functions of other tissues are likely compromised at the expense of maintenance of the mucus layer in mucin-producing tissues.