Glycerol-3-phosphate acyltransferase (GPAT) catalyzes the first committed step of de novo triacylglycerol synthesis by converting glycerol-3-phosphate to lysophosphatidic acid (LPA). LPA is a mitogen that mediates multiple cellular processes including cell proliferation. Four GPAT isoforms have been cloned to date. GPAT4 is strongly expressed in the mouse testis. Reverse transcription- polymerase chain reaction (PCR), real-time PCR, and in situ hybridization (ISH) were used to analyze the GPAT4 expression and to localize the expressing cell types in the mouse testis during postnatal development. GPAT4 cDNA was inserted into pcDNA4/His to construct a recombinant vector, which was transfected into a mouse spermatogonial cell line (GC-1spg). GPAT4 was first expressed in mice at 2 weeks postnatally. Expression was abundant from the third week, plateaued at week 5-6 and then maintained at a high level in the adult. ISH revealed that GPAT4 gene was expressed abundantly in spermatocytes and around spermatids during meiosis but not in elongated spermatids during later spermiogenesis. GC-1spg cells showed a marked increase in proliferation after transfection with GPAT4; cell cycle analysis showed a decrease in the percentage of cells in the G0/G1 phase and an increase in the S phase. Thus, GPAT4 might play an important role in spermatogenesis, especially in mid-meiosis.