Total lipid extracts were obtained from SH-SY5Y human neuroblastoma cells grown to confluency in mycoplasma-free 10% fetal calf serum. The major glycerophospholipid classes and free diacylglycerols (DAG) were isolated and quantitated by silicic acid and DEAE-cellulose column and thin-layer chromatography. The choline (CGPL), ethanolamine (EGPL), serine (SGPL), and inositol (IGPL) glycerophospholipids were converted to the corresponding diradylglycerols by phospholipase C. The molecular species of the diradylglycerols were determined by capillary gas-liquid chromatography of the trimethylsilyl or t-butyldimethylsilyl ethers. The CGPL was rich in the oligoenoic species and IGPL was rich in the polyenoic species, especially the 18:0-20:4(n-6). The EGPL contained 30-40% diacyl, 60-64% alkenylacyl, and 1-3% alkylacyl species, which were also rich in polyenoic derivatives. Small amounts of alkenylacyl species were detected also in CGPL. The cellular DAG possessed a molecular species composition halfway between those of the DAG moieties of CGPL and IGPL. The cells grown in the presence of 10% calf serum exhibited great variability in the content of 20:3(n-9) fatty acid, which was found to substitute for the 20:4(n-6) acid in the molecular species with 18:0 in both IGPL and DAG. The 20:3(n-9) was largely absent from the CGPL, but occurred also in EGPL, where it was preferentially associated with 18:0 in the diacyl but with 18:1 in the alkylacyl and alkenylacyl species. The detailed documentation of molecular species of glycerophospholipids of the neuroblastoma cells offers new opportunities for identification of the source of free DAG released in transmembrane signalling.