The diets of laboratory rats were isotopically and nutritionally manipulated using purified C3 and/or C4 macronutrients to investigate the routing of dietary carbon to bone collagen biosynthesis. Diets were formulated with purified proteins, carbohydrates and lipids of defined composition and natural abundance stable isotope ratios. Bulk protein and constituent amino acid delta(13)C values determined for whole diet and bone collagen provided the basis for assessing isotopic fractionation and estimating the degree of routing versus synthesis de novo of essential, non-essential and conditionally indispensable amino acids. Essential and conditionally indispensable amino acids were shown to be routed from diet to collagen with little isotopic fractionation whereas non-essential amino acids differed by up to 20 per thousand. Mathematical modelling of the relationships between macronutrient and tissue delta(13)C values provided qualitative and quantitative insights into the metabolic and energetic controls on bone collagen biosynthesis. Essential amino acids comprise 21.7 % of the carbon in collagen, defining the minimum amount of dietary carbon routing. Estimates of 42 and 28 % routing were shown for the non-essential amino acids, glycine and aspartate, respectively. In total, the routing of non-essential and conditionally indispensable amino acids was estimated to equal 29.6 % of the carbon in collagen. When the contribution of carbon from the essential amino acids is also considered, we arrive at an overall minimum estimate of 51.3 % routing of dietary amino acid carbon into bone collagen.