In order to study the lipid composition of human white matter, we have developed a 31P NMR spectroscopy method, which allows the determination and quantitation of the main phospholipids found in biological membranes. The technique is based upon the use of a non-ionic detergent (Triton X-100) which induces, in aqueous media, the formation of mixed micelles that are magnetically isotropic. The linewidths and chemical shifts depend on both the molar ratio detergent/phospholipid and the pH of the suspension. After determination of the optimum values for these two parameters, 31P NMR spectra were recorded, in which all phospholipid resonances were resolved. After determining precise chemical shifts for each phospholipid, concentrations were measured by comparing the peak areas with that of an internal standard. Analysis of the complex phospholipid composition of human white matter using this method gave values very close to that found in the literature for such tissue. Moreover this nondestructive method proved to be very sensitive since less than 1 mg of a mixture of phospholipids was needed.