Pigs were reared in laboratory pens on controlled diets that consisted of either 100% C₃ plants or 100% C₄ plants. Carbon and nitrogen isotopic compositions of the diets, and the resulting pig products, purified collagen and muscle tissue, were measured to determine isotopic fractionation during growth and metabolism. Total collagen from pigs grown on C₃ diets was enriched in ¹³C by 3.2per thousand and in ¹⁵N by 2.2per thousand, whereas that from pigs reared on C₄ diets was enriched in ¹³C by 1.4per thousand and in¹⁵N by 2.3per thousand. In addition, fractionation between pigs and their diets was determined at the molecular level on individual amino acids separated by ion exchange chromatography. The carbon isotopic compositions of separated amino acids from the C₃ and C₄ diets were transferred to amino acids in bone collagen. For nitrogen, the isotopic compositions of all nonessential amino acids were enriched in ¹⁵N relative to those amino acids in the diet. Threonine, an essential amino acid, behaved oppositely, in that its isotope ratio (delta¹⁵N) was depleted by an average of 6per thousand from the delta¹⁵N of the whole collagen. Similar isotopic patterns were analyzed in collagenous amino acids extracted from field specimens that included both herbivores and carnivores; marine animals and terrestrial animals; and C₃ and C₄ feeders. Amino acids from two fossil bones, a bison (4,500 years old) and a whale (70,000 years old), recorded the same isotopic signals as modern collagen. The ubiquity of these isotopic patterns at the molecular level suggests that distinct biochemical mechanisms control the metabolism of amino acids in animals rather than random synthesis.