Identification of transcription factors involved in the progression of spermatogenic cell differentiation is important for understanding the molecular mechanisms controlling spermatogenesis. To this end, we utilized the mouse SP-10 gene encoding a conserved acrosomal protein as an experimental model. Promoter analysis in transgenic mice had previously shown that the -186/-91 region of the SP-10 promoter was critical for spermatid-specific expression. Here, we focus on a purine (Pu) box (-agaaaa) located at -154, which is conserved in the mouse, monkey, and human SP-10 gene promoters. NF45 and NF90, which belong to the family of nuclear factor of activated T cells (NFAT), are known as Pu-box-binding proteins. We tested the potential of NF45 and NF90 to activate the SP-10 promoter via the Pu-box element. Immunohistochemistry showed the presence of NF45 and NF90 in the nuclei of pachytene spermatocytes, round spermatids, and Sertoli cells. In gel shift assays, recombinant NF45 bound to the mouse SP-10 promoter in an AGAAAA site-specific manner. Cotransfection of NF45 and NF90 up-regulated SP-10 promoter-driven luciferase expression in transiently transfected spermatogenic GC2 cell line; this up-regulation required the -AGAAAA- site. Furthermore, stimulation of the endogenous NF45-NF90 complex in Jurkat cells by phorbol myristate acetate + ionomycin up-regulated the SP-10 promoter activity in plasmid-based assays. In the context of chromatin, however, stimulation of NF45-NF90 alone was not sufficient to activate an SP-10 promoter-driven green fluorescent protein transgene. Based on these results, we propose that NF45 and NF90 have the potential to activate SP-10 gene transcription, and that a chromatin modification event must occur first in order to provide access to these transcription factors.