Abstract: The aim of present study was to characterize nitric oxide production via nitrite measurement as its stable metabolite in the different species of spermatozoa (human, bull and ram). Sperm samples were fractionated on the basis of density with the use of a three-layer Percoll gradient. Spermatozoa collected from the various percoll gradient interfaces (50/70 and 70/95%). Results showed that nitrite is produced by human, bull and ram spermatozoa, but in the same sample, there were no significant difference between high density (with higher percentage of motility) sperm subpopulation and low density (with lower percentage of motility) sperm subpopulation Therefore, it seems that spermatozoa with different motility in the one sperm population have the same production of NO in physiological condition that is different from pathological condition (e.g., in the subfertile men).