The binding of capacitated spermatozoa to the egg's extracellular coat and induction of acrosome reaction are necessary for successful fertilization in mammals. Biogenesis of acrosome is complicated, and not all proteins involved in this process are known. In this study, we have cloned a novel mouse gene, Spaca7, that is expressed exclusively in the testes. During the postnatal development, transcripts of the gene could be detected at a very low level in 18-day-old mouse testes and at a higher level in 21-day-old mouse testes and later, which corresponds to an expansion of round spermatids. In the stably transfected PT67 cells, SPACA7 fused with EGFP was predominantly localized in the Golgi apparatus. In transgenic mouse testes, the fusion protein was found in acrosome (starting from the first stages of acrosome formation in late pachytene spermatocytes and finally in spermatozoa isolated from caput and cauda of epididymis). Confocal microscopy studies revealed an intra-acrosomal not membrane-bound localization of SPACA7/EGFP, which suggests that the protein can be released during acrosome reaction and involved in fertilization. Acrosomal localization of endogenous SPACA7 protein was also found in human spermatozoa.