Fast atom bombardment (FAB) mass spectrometry was evaluated as a means for the characterization of molecular species of glycerophosphocholines (GPC) from HL60 cells. Previous reports of phospholipid molecular species analysis have suggested that several inherent problems with FAB could limit its analytical usefulness in such an application. The GPC-related secondary ions produced by the FAB experiment were found to be dependent on the matrix employed. Triethanolamine was found to minimize mass-related discrimination in ion emission when mixtures were studied; and furthermore, this matrix maintained 60% maximal ion current after 12 minutes as compared to a glycerol matrix which diminished to 10% maximal ion current. Using triethanolamine, the major GPC species in HL60 cells prelabeled with (2H8) arachidonic acid were found to be 16:0a16:1, 16:0e/18:1, 16:0a/18:1, 18:1a/18:1 and 18:0a/18:1. It was possible to identify the minor GPC species containing arachidonic acid only after partial purification by reverse-phase high-performance liquid chromatography. Comparison of the 2H8/2H0 enrichment data estimated by FAB with data obtained by gas chromatographic/mass spectral analysis of arachidonic acid following GPC hydrolysis revealed that the precision of FAB was less precise than gas chromatography/mass spectrometry. Yet the FAB technique did allow the observation of one unexpected molecular species (18:1a/20:4) due to the fact that the GPC was not degraded to simpler species prior to analysis. In this respect, the two strategies of molecular species analysis complement each other.