A nondestructive method has been developed for rapid analysis of lipid content of membrane extracts based on high field proton NMR spectroscopy. Lipid extraction is done by stepwise sonication of purified membranes into chloroform:methanol:water mixtures, and 1H spectra are recorded at 35 degrees C on final preparations consisting of at least 1 mg dried lipid solubilized in 2:1 CD3OD:CDCl3. Spectral peaks of lipid mixtures are assigned to lipid classes using a data base of standard lipid characteristic resonances derived from purified single membrane lipids and known mixtures of them. Peak intensities of characteristic peaks yield ratios of various lipids such as cholesterol:phospholipid and phosphatidylcholine:phosphatidylethanolamine, degree of unsaturation, average acyl chain length, total glycerol lipid content, and presence or absence of particular lipids, such as glycolipids or lysolipids. This procedure of membrane lipid analysis has been verified using known mixtures of purified standard lipids.