Sphingomyelin synthase 2 (SMS2) is an enzyme that catalyzes the conversion of phosphatidylcholine and ceramide to sphingomyelin and diacylglycerol, and it is crucial to cellular lipid metabolism. Using the technique of subtraction hybridization, we have isolated a full-length cDNA encoding SMS2 from rat testes, which shared 93 and 87% identity at the nucleotide level with SMS2 in mice and humans respectively. A specific polyclonal antibody was prepared against a 20 amino acid peptide of NH2-FSWPLSWPPGCFKSSCKKYS-COOH near the C-terminus of SMS2. Studies by RT-PCR and immunoblotting have shown that the expression of SMS2 was limited to late round spermatids and elongating spermatids, but it was not detected in late elongate spermatids and Sertoli cells. Furthermore, SMS2 was shown to associate with the developing acrosome beginning in late round spermatid through elongating spermatids (but not late elongate spermatids) and the cell membrane in studies using fluorescent microscopy and immunohistochemistry. These data were further confirmed by studies using immunogold electron microscopy. The expression of SMS2 in the seminiferous epithelium is stage-specific with its highest expression detected in the acrosome region in late round spermatids from stages VIII–IX, and also in the acrosome in elongating spermatids with diminished intensity in stages X–V; however, it was not found in the acrosome in elongate spermatids in stages VI–VIII. Collectively, these results suggest that SMS2 may play a crucial role in the lipid metabolism in acrosome formation and the plasma membrane restructuring from late round spermatids to early elongating spermatids.