A number of recent studies have attempted to trace diet at different stages of an individual's life by comparing isotope ratios of bone from different gross anatomical sites within the skeleton. In this study we develop this approach further by separating bone of differing mineral densities within one skeletal element, where each density fraction represents a different period of time. Isotope ratios are measured for these fractions. Each density fraction represents a period of bone formation and maturation, where younger (more recently formed) bone is less well-mineralized and therefore less dense than relatively older packets of bone. In an adult, bone is therefore able to partition approximately the last 15 years of life. Bone fractions were recovered by stepped ultracentrifugation in a series of organic solvents of increasing density, and then collagen was recovered by decalcification in dilute acid, and stable carbon isotope ratios (13C/12C) were measured. Bone density microstructure was checked for bacterial remodelling using backscattered electron imaging in a scanning electron microscope. Our results indicate that the bone density fractionation method is applicable to archaeological material, here extending to a maximum of 5,000 years BP, and that collagen can successfully be extracted from such fractions. The carbon isotope values for bone fractions of different densities patterned out as expected in one modern control bone and in specimens from five archaeological human skeletons, including three precolonial hunter-gatherers and two 18th/19th century individuals. The latter two are known (from previous assessments) to have undergone marked changes in diet during their lifetimes. Postmortem alteration was evident in some of the specimens examined. The bone density fractionation approach has allowed greater resolution of diet than has hitherto been possible and has provided access to the elusive last years and months of an individual's life. Am J Phys Anthropol 116:66–79, 2001. © 2001 Wiley-Liss, Inc.