Amino acid analysis of organic solvent extracts of tissue material has been evaluated for determination of protein content. Conventional ninhydrin-based analysis does not allow determination of a large number of lipid-rich samples. Therefore, the hydrolyzed samples were treated with phenylisothiocyanate and the phenylthiocarbamyl (PTC) derivatives obtained were separated by reverse-phase HPLC. With this method, analysis of many lipid-rich samples is feasible. In addition, phosphatidylethanolamine and phosphatidylserine can then be determined together with the amino acid constituents. The PTC/reverse-phase HPLC method was used for analysis of chloroform/ methanol extracts of spinal cord, lung, and bile after chromatography on Lipidex 5000 in methanol/ethylene chloride, 4:1 (v/v). The chromatography profiles show that in all tissue samples the proteins elute before the phospholipids. Consequently, a single step of Lipidex 5000 chromatography can be used to purify polypeptides present in organic solvent extracts. Using pulmonary surfactant extracts (with about 98% phospholipids and 1-2% proteins), we find that individual contents of surfactant proteins B and C can be determined by amino acid analysis.