Prolyl oligopeptidase (POP) expression in mouse testis during sexual maturation was examined. Northern blot analysis showed that POP mRNA expression was highest at 2 weeks of age, and gradually reduced thereafter. However, enzyme activity was almost constant during the examined period. In situ hybridization study revealed a change in the expression site of POP mRNA in testis during sexual maturation. Positive signals were detected in all types of cells in the seminiferous tubules before maturation, and were restricted to spermatids at the spermatogenesis cycle stages I-VIII in adult mice. POP was detected in the insoluble fraction of sperm by Western blot analysis. Immunohistochemical analyses showed that POP is localized in the spermatids at steps 12–16 of spermiogenesis and in the midpiece of the sperm fragellum. It was also found that specific POP inhibitors, poststatin and benzyloxycarbonyl-pro-line-prolinal, suppressed sperm motility. These results suggest that POP may be involved in meiosis of spermatocytes, differentiation of spermatids, and sperm motility in the mouse.