Quantitative one-dimensional (1D) H-1 NMR spectroscopy is a useful tool for determining metabolite concentrations because of the direct proportionality of signal intensity to the quantity of analyte. However, severe signal overlap in 1D H-1 NMR spectra of complex metabolite mixtures hinders accurate quantification. Extension of 1D H-1 to 2D H-1-C-13 HSQC leads to the dispersion of peaks along the C-13 dimension and greatly alleviates peak overlapping. Although veaks are better resolved in 2D H-1-C-13 HSQC than in ID H NMR spectra, the simple proportionality of cross peaks to the quantity of individual metabolites is lost by resonance-specific signal attenuation during the coherence transfer periods. As a result, peaks for individual metabolites usually are quantified by reference to calibration data collected from samples of known concentration. We show here that data from a series of HSQC spectra acquired with incremented repetition times (the time between the end of the first H-1 excitation pulse to the beginning of data acquisition) can be extrapolated back to zero time to yield a time-zero 2D H-1-C-13 HSQC spectrum (HSQC(0)) in which signal intensities are proportional to concentrations of individual metabolites. Relative concentrations determined from cross peak intensities can be converted to absolute concentrations by reference to an internal standard of known concentration. Clustering of the HSQC(0) cross peaks by their normalized intensities identifies those corresponding to metabolites present at a given concentration, and this information can assist in assigning these peaks to specific compounds. The concentration measurement for an individual metabolite can be improved by averaging the intensities of multiple, nonoverlapping cross peaks assigned to that metabolite.