Very long chain (VLC) PUFA of the n-6 and n-3 series are known to occur in mammalian testis. The aim of this work was to characterize further two testicular lipid classes with VLCPUFA, cholesterol esters (CE) and total triglycerides (TG) in rat and mouse testis. The VLCPUFA predominating in these lipids were a series of n-6 pentaenes and tetraenes with 24 to 32 carbons, including small amounts of odd-chain PUFA, 28:5n-6 and 24:5n-6 prevailing in CE and TG, respectively. Most of the VLCPUFA of TG were concentrated in a small fraction of TG, made up by 1-O-alkyl-2,3-DAG. This TG subclass was absent altogether from the TG of sexually immature testis. The TG and the CE with VLCPUFA only occurred in testis of adult fertile animals. The proportion of VLCPUFA in total TG and CE was higher in rodents than in other mammals. In the n-6 PUFA-rich adult mouse testis, the amounts of testicular triacylglycerols decreased significantly after consumption of fish oil for 2 wk. Whereas 18:2n-6 was significantly reduced, the amounts of 22:5n-6 and longer n-6 PUFA were less affected in all major testicular lipids including PC and PE, where they were unchanged. The 1-O-alkyl-2,3-DAG and their n-6 VLCPUFA were virtually unaffected by the diet. The VLCPUFA-containing molecular species of CE and TG may represent a form of storage of cholesterol and polyenoic FA required to sustain spermatogenesis. Via chain-shortening, VLCPUFA stored in the neutral lipids may serve as precursors of the major C22 PUFA typical of cell membrane glycerophospholipids, protecting testicular cells against shifts in FA composition induced by dietary changes.