Isotopic studies of palaeoecological and ecological questions often use bone collagen or bioapatite as substrates, but rarely both. Substantial new information can be gained from the incorporation of isotopic values from both the organic and inorganic fractions of bone. Here we show that combining isotopic data from both substrates provides valuable and unique insights into (1) trophic relationships and dietary interactions; (2) differences in digestive physiologies and (3) identification of palaeontological or archaeological remains that lack diagnostic morphological characters. We present a range of new isotopic data collected from modern and fossil mammals, and investigate patterns within several published datasets. We define carbon isotope spacing variables, and then explore four diverse palaeoecological and ecological case studies. Copyright © 2009 John Wiley & Sons, Ltd.