The lipids of the hepatic tumor cell line, PLHC-1, from the topminnow (Poeciliopsis lucida), were found to contain considerable amounts of a range of n-9 polyunsaturated fatty acids despite culture in serum containing significant amounts of essential fatty acids. The structural identity of all the n-9 polyunsaturated fatty acids was confirmed by gas chromatography/mass spectrometry. Of particular interest, PLHC-1 cell total lipid contained 1.9% of 22:3n-9 and 3.3% of 22:4n-9. As the culture medium contained virtually no n-9 polyunsaturated fatty acids, these fatty acids are all formed by the PLHC-1 cells, presumably from 18:1n-9. The 22:3n-9 and 22:4n-9 are presumably formed by processes of elongation and "delta 4" desaturation of Mead acid, 20:3n-9, present at over 11% in fatty acids of total lipid. Both 22:3n-9 and 22:4n-9 were primarily located in phosphatidylserine (4.1 and 8.5%, respectively) and, to a lesser extent, in phosphatidylethanolamine (2.2 and 6.5%, respectively), in common with the C22 derivatives of the n-3 and n-6 series, whereas 20:3n-9 was preferentially located in phosphatidylinositol (31.2%). The results establish that long-chain polyunsaturated fatty acids of the n-9 series can be formed in vertebrate tissue other than in conditions of classical essential fatty acid deficiency.