Sertoli cells supply germ cells with nutrients, including highly polyunsaturated fatty acids (hPUFAs), which are essential for testicular function. We have previously reported high expression of lysophosphatidic acid acyltransferase (LPAAT)3 in mature mouse testis and suggested an arachidonoyl-transferase activity to LPA. To investigate the role of LPAAT3 in the storage and release of PUFAs, TM4 Sertoli cells were stably transfected with LPAAT3-small hairpin (sh)RNA. Arachidonoyl-, eicosapentaenoyl-, and docosapentaenoyl-containing phosphatidylcholine (PC) and linoleoyl-containing phosphatidylethanolamine (PE), phosphatidylserine (PS), and phosphatidylglycerol were significantly decreased as determined by liquid chromatography coupled to electrospray ionization mass spectrometry. Expression of murine LPAAT3 in Chinese hamster ovary (CHO)-K1 cells had essentially an opposite effect. The level of polyunsaturated PC correlated with cellular levels of free docosapentaenoic acid and eicosapentaenoic acid in TM4 and CHO-K1 cells, respectively. Activity assays using microsomal preparations as a source of LPAAT3 revealed an excessive PA synthesis from LPA acceptors for docosahexaenoyl-, arachidonoyl- and less pronounced for linoleoyl-CoA. We propose that the efficient incorporation of hPUFAs into PA-the precursor of several phospholipids, including PC-and the selective increase of the polyunsaturated PC pool in TM4 Sertoli cells might be required for the controlled release of hPUFAs and their supply to germ cells.