Objective: We sought to compare endothelial nitric oxide synthase (eNOS) expression in sperm from normospermic and infertile males. Design: Prospective, observational study. Materials/Methods: Twenty-nine diagnostic semen specimens (15 normospermic, 6 multiple male-factor, 4 teratospermic, 4 asthenospermic), and 4 epididymal aspirations were used in this study. Motile sperm was selected by swim-up. For Western blot analysis, sperm proteins were separated by 1D-PAGE, 12% (20 μg protein/lane), and transferred to nitrocellulose sheets. The resulting Western blot was probed with polyclonal antibodies to eNOS and detection was accomplished using a biotinylated secondary antibody, a streptavidin-horseradish peroxidase complex, and enhanced chemiluminescence. Indirect immunofluorescence was accomplished using antibodies to eNOS and FITC-conjugated secondary antibody, and examined with an Olympus fluorescence microscope. Samples were characterized as normospermic, asthenospermic, teratospermic and epididymal. Endothelial NOS activity was examined by measuring nitrite, the stable end-product of NOS, using the Griess reaction. Our main outcome measures were localization of eNOS on human sperm and correlation with sperm characteristic (normospermic, teratospermic, asthenospermic, and epididymal), and comparison of eNOS activity in sperm from normospermic and infertile males. Results: Western blot analysis showed the presence of eNOS as a band of 135-140 kDa in both donor’s and infertile patient’s sperm. However, nitrite production in sperm cultures from asthenospermic males was nearly twenty-fold compared to sperm cultures from normospermic males (1.368 mM and 0.073 mM, respectively, P < 0.05). In addition, immunocytochemical analysis of eNOS showed distinct staining patterns on sperm. Staining patterns on asthenospermic specimens compared closely to normospermic, while teratospermic and epididymal were quite different. The most prevalent staining pattern for normospermic specimens was staining on both midpiece and equatorial regions, occurring in 70.5% of sperm scored. This compares to 54.0% for asthenospermic, 18.0% for teratospermic, and 8.9% for epididymal sperm. The majority of staining on teratospermic specimens was on midpiece only (43.3% of sperm scored), while 18.4% of sperm scored had no staining. On the other hand, the major patterns of staining for epididymal specimens was no staining on 28.8% of sperm scored, and equatorial only on 23.9% of sperm scored. Some epididymal sperm had a unique staining pattern not found on any of the other specimens. This was a speckled pattern occurring on the perimeter of the head only, and was observed in 12.5% of the sperm scored. Conclusions: The present study provides compelling evidence that eNOS is present in the sperm of both fertile and infertile males. Although the eNOS immunostaining patterns of normospermic and asthenospermic samples were similar, nitrite accumulation was significantly higher for asthenospermic samples suggesting a physiological role for nitric oxide in the regulation of sperm motility. Furthermore, the distinct eNOS immunostaining pattern in epididymal samples suggests that nitric oxide may also be involved in the regulation of sperm maturation. Supported By: Minority Biomedical Research Support Grant #GM08037-27 and SCORE Grant #2SO6GMO8037-28.