Purpose of reviewOur goal is to explain how2H2O, a stable isotope of water, can be used to quantify metabolic fluxin vivo. Although2H2O was originally used more than 65 years ago, recent reports have described new applications. Investigators have also refined the original work. We discuss those points, emphasizing factors that should be considered when interpreting data. As our goal is to discuss why2H2O is a novel tracer for studying metabolism, we do not comment on the interpretation(s) of physiological data.Recent findingsIt is clear that2H2O can now be used to address questions related to carbohydrate, lipid, protein and DNA synthesis. A particular advantage of using2H2O is that investigators can study metabolism in free-living individuals. Using this novel tracer method, it is thus possible to elucidate new, highly relevant, knowledge regarding health and disease.SummaryAs the use of2H2O requires few assumptions, the application of this tracer should yield sound information regarding the regulation of biochemical reactionsin vivo.