Stable isotope analysis has become an essential tool in investigations of ancient migration and paleodietary reconstruction. Because the biogeochemistry of bone collagen and apatite is well known, current methods rely almost exclusively on analyses of bones and teeth; however, dental calculus represents a potentially additional biological source of isotopic data from ancient skeletons. Dental calculus is a mineralized bacterial biofilm that forms on the surfaces of teeth. Sampling dental calculus does not damage the dentition and thus can be used in cases where it is not possible to perform destructive analyses of conventional mineralized tissues. Like bone and dentine, dental calculus contains both inorganic and organic components, allowing measurement of C, N, O, H, and Sr isotopes. Additionally, dental calculus forms as serial, non-remodeling laminar accretions on the tooth surface, opening up the possibility of analyzing discrete time points during the lifetime of an individual. However, as a microbial biofilm and not a human tissue, the biochemistry of dental calculus is complex, containing multiple calcium phosphate mineral phases, organic and inorganic food remains, hundreds of human and bacterial proteins, and diverse biomolecules from thousands of endogenous bacterial taxa. Isotopic investigation of dental calculus is still in its infancy, and many questions remain regarding its formation and processes of diagenesis. This chapter (1) reviews the unique advantages presented by dental calculus as a novel source of biological isotopic data, (2) critically evaluates published isotopic studies of dental calculus, and (3) explores the current challenges of dental calculus stable isotope analysis through a case study of an Ancient Puebloan Basketmaker II population from the American Southwest.