We are developing a method for quantifying plasma lipoproteins by proton nuclear magnetic resonance (NMR) spectroscopy that offers advantages over existing clinical methods. We showed that the major lipoproteins have distinct NMR properties sufficient to permit their concentrations to be extracted from a computer lineshape analysis of the plasma lipid methyl resonance envelope (Clin Chem 1991; 37:377-86). We have now discovered that the spectra of the subspecies within each lipoprotein class are different enough to influence the composite spectrum of that class and hence the spectrum of whole plasma. By including spectra representative of these subspecies as additional components in the lineshape-fitting analysis, a complete concentration profile of very-low-density, low-density (LDL), and high-density (HDL) lipoproteins plus the subspecies distributions within these classes can be simultaneously generated. A pilot study of 30 plasma samples of widely varied composition demonstrated good agreement between NMR-derived values and lipoprotein lipid concentrations and LDL and HDL subspecies distributions determined by gradient-gel electrophoresis.