Isotopically labeling a metabolite and tracing its metabolic fate has provided invaluable insights about the role of metabolism in human diseases in addition to a variety of other issues. C-labeled metabolite tracers or unlabeledH-based NMR experiments are currently the most common application of NMR to metabolomics studies. Unfortunately, the coverage of the metabolome has been consequently limited to the most abundant carbon-containing metabolites. To expand the coverage of the metabolome and enhance the impact of metabolomics studies, we present a protocol for N-labeled metabolite tracer experiments that may also be combined with routineC tracer experiments to simultaneously detect both N- andC-labeled metabolites in metabolic samples. A database consisting of 2D H-N HSQC natural-abundance spectra of 50 nitrogen-containing metabolites are also presented to facilitate the assignment of N-labeled metabolites. The methodology is demonstrated by labeling Escherichia coli and Staphylococcus aureus metabolomes withN-ammonium chloride, N-arginine, and C-acetate. Efficient N andC metabolite labeling and identification were achieved utilizing standard cell culture and sample preparation protocols.