The relative distribution of intact diacylphosphatidylcholine species isolated from the lung lavage fluid of rabbits has been investigated by positive ion fast-atom bombardment (FAB) mass spectrometry. Two different isolation/purification methods were applied and evaluated prior to mass spectrometric analysis. The first method consisted of a Bligh and Dyer extraction of the lung lavage fluid followed by isocratic high-performance liquid chromatographic (HPLC) separation. In the second method a thin-layer chromatographic purification step was introduced between the extraction procedure and the HPLC separation. Further, the FAB matrices glycerol and 3-nitrobenzyl alcohol were used, and their influence on the diacylphosphatidylcholine molecular ion species was studied. The Bligh and Dyer extraction followed by the simple HPLC separation was the method of choice to obtain stable, long-lasting protonated molecular ions and diagnostic fragment ions, which permitted the identification of the polar head-group. In combination with 3-nitrobenzyl alcohol as liquid matrix we established a procedure that yielded a fast sample preparation method, a good signal-to-noise ratio for detecting minor species, and reduced formation of [M+H-2H]+ ion species. The relative fatty acid composition of the diacylphosphatidylcholine fractions isolated from rabbit lung lavage fluid was determined by negative ion FAB mass spectrometry using the carboxylate anions. The mass spectrometric results were compared with those acquired by gas chromatographic determination of the fatty acid methyl esters. Close agreement was found between the data obtained by the two independent methods.