We report the first chemoenzymatic synthesis of the stable isotope-enriched heparin from a uniformly labeled [(13)C,(15)N]N-acetylheparosan (-GlcA(1,4)GlcNAc-) prepared from E. coli K5. Glycosaminoglycan (GAG) precursors and heparin were formed from N-acetylheparosan by the following steps: chemical N-deacetylation and N-sulfonation leading to N-sulfoheparosan (-GlcA(1,4)GlcNS-); enzyme-catalyzed C5-epimerization and 2-O-sulfonation leading to undersulfated heparin (-IdoA2S(1,4)GlcNS-); enzymatic 6-O-sulfonation leading to the heparin backbone (-IdoA2S(1,4)GlcNS6S-); and selective enzymatic 3-O-sulfonation leading to the anticoagulant heparin, containing the GlcNS6S3S residue. Heteronuclear, multidimensional nuclear magnetic resonance spectroscopy was employed to analyze the chemical composition and solution structure of [(13)C,(15)N]N-acetylheparosan, precursors, and heparin. Isotopic enrichment was found to provide well-resolved (13)C spectra with the high sensitivity required for conformational studies of these biomolecules. Stable isotope-labeled heparin was indistinguishable from heparin derived from animal tissues and is a novel reagent for studying the interaction of heparin with proteins.