Dietary deficiency of essential fatty acids of the n−3 and n−6 series is known to promote a compensatory increase in polyenoic fatty acids of the n−9 series in the lipids of mammalian tissues. In the present study long-chain n−9 polyenes were found to be normal components of the epididymis and especially of sperm isolated from that tissue, in healthy, well-fed, fertile rats maintained on essential fatty acid-sufficient diets. The n−9 polyenes occurred in large concentrations in the choline glycerophospholipids (CGP), the major phospholipid class of spermatozoa in epididymalcauda, and were highly concentrated in plasmenylcholine, the major subclass of CGP. The uncommon polyene 22∶4n−9 was found in the highest proportion, followed in order of relative abundance by 22∶3n−9, 20∶3n−9 and 24∶4n−9. These polyenes were probably derived from oleate (18∶1n−9) in much the same way as long-chain polyenes of the n−6 and n−3 series are derived from linoleate (18∶2n−6) and linolenate (18∶3n−3), respectively.