The polyol metabolizing pathway, which consists of two enzymes, aldose reductase (AR) and sorbitol dehydrogenase (SDH), converts glucose to fructose. The enzymatic activities, expression, and localization of AR and SDH were studied in reproductive tracts and spermatozoa of male rats by immunohistochemistry, Western blotting, and enzyme assays. Immunoreactivity to an AR antibody was observed mainly in epithelia of epididymis, seminal vesicle, vas deferens, and prostate gland in adult rats. Similar staining profiles were observed for these tissues when an SDH antibody was used. However, in testis, the cells that express these 2 enzymes differed; whereas AR was expressed in Sertoli cells and to lesser extent in spermatogenic cells, SDH was detected in spermatogenic cells of seminiferous tubules. This cell type‐specific gene expression was confirmed in primary cultured cells isolated from rat testes. SDH protein levels were higher during spermatid elongation, and large amounts of SDH were carried over to the spermatozoa. Because one of the functions of members of the aldo‐keto reductase superfamily is to detoxify harmful carbonyl compounds, an intrinsic function of AR in Sertoli cells may be to catalyze the reduction of cytotoxic metabolites, such as lipid peroxidation products and steroid hormones, which are produced during spermatogenesis. Because uterine fluid and seminal plasma both contain sorbitol, it is likely that SDH in spermatozoa converts sorbitol to fructose for use as an energy source.