This research reported in this paper investigated the relationship between diet and bone FA and cholesterol in rats raised on a variety of isotopically controlled diets comprising 20% C3 or C4 protein (casein) and C3 and/or C4 nonprotein or energy (sucrose, starch, and oil) macronutrients. Compoundspecific stable carbon isotope analysis (δ13C) was performed on the FA (16∶0, 18∶0, 18∶1, and 18∶2) and cholesterol isolated from the diet (n=4) and bone (n=8) of these animals. The dietary signals reflected by the bone lipids were investigated using linear regression analysis. δ13C values of bone cholesterol and stearic (18∶0) acid were shown to reflect whole-diet δ13C values. whereas the δ13C values of bone palmitic (16∶0), oleic (18∶1), and linoleic (18∶2) acids reflected dietary FA δ13C values. Dietary signal differences are a result of the balance between direct incorporation (or routing) and de novo synthesis of each of these bone lipids. Estimates of the degree of routing of these bone lipids gleaned from correlations between Δ13C dlipid-wdiet (δ13Cdiet lipid-δ13Cwhole diet) spacings and Δ13C blipid-wdiet (δ13Cbone lipid-δ13Cwhole diet fractionations demonstrated that the extent of routing, where 18∶2>16∶0>18∶1>18∶0>cholesterol, reflected the relative abundances of these lipids in the diet. These findings provide the basis for more accurate insights into diet when the δ13C analysis of bone fatty FA or cholesterol is employed.