Aquaporin-1 (AQP-1), a six-transmembrane domain protein, is found to be responsible for water transport. The purpose of this study was to investigate the expression of AQP-1 mRNA and protein in the testis, epididymis, vas deferens, ventral prostate, and seminal vesicle from mature mice by using reverse transcription polymerase chain reaction (RT-PCR) and Western blotting, and the cellular localization of AQP-1 by immunohistochemistry. RT-PCR revealed that AQP-1 mRNA was expressed in all organs we examined. Western blotting displayed a 29-kDa band and a 35- to 45-kDa band corresponding to non-glycosylated and/or glycosylated AQP-1 in those organs. The immunohistochemical evidence showed that AQP-1 was mainly located on the plasma membrane of epithelial cells of the rete testis, vas deferens, ventral prostate, seminal vesicle and the non-ciliated cells of the proximal and distal efferent ducts. However, AQP-1 was absent from spermatogenic cells lining the seminiferous epithelium and from the spermatozoa in the lumen of the distal efferent duct. These findings provide valuable information on the expression of AQP-1 in male reproductive organs and suggest that AQP-1 is involved in water transport to regulate water homeostasis in male reproductive physiology.