Spermiogenesis is the terminal phase of male germ cell differentiation during which haploid spermatids engage in coordinate expression of a number of testis-specific genes, including those specifying acrosomal proteins. To begin to understand the transcriptional regulation during acrosomal biogenesis, we initiated promoter analysis of the gene encoding the acrosomal protein SP-10. SP-10 was previously shown to be transcribed within Golgi-phase round spermatids in the human. The present study characterizes SP-10 gene expression during spermiogenesis in the mouse and identifies regions of the mouse SP-10 (mSP-10) promoter that are capable of driving round spermatid-specific transcription in vivo. Expression of mSP-10 mRNA was initiated in early round spermatids coincident with acrosomal biogenesis and was terminated prior to nuclear elongation. The core promoter of mSP-10 lacked a TATA box but contained a canonical initiator (Inr) element surrounding the transcription start site. Using transgenic mice, we showed that the -408 to +28-base pair (bp) or the -266 to +28-bp mSP-10 5' flanking region is sufficient to direct round spermatid-specific expression of a green fluorescent protein reporter gene. On the other hand, the -91 to +28-bp mSP-10 gene fragment lacked promoter activity in vivo. This is the first functional characterization of a testis-specific gene promoter active in early round spermatids.