The testicular isozyme of angiotensin-converting enzyme (ACE) is associated with male fertility. Spermatozoa from mice lacking ACE showed defects in transport within the oviducts and in binding to zonae pellucidae although the animals had normal sperm count, morphology and motility. In fact, unexplained infertility is difficult to be predicted by conventional parameters such as sperm count. We measured membrane testicular ACE activity in a sperm suspension in PBS and total testis ACE activity in spermatozoa by solubilization with Triton X-100. Total testis ACE activity and membrane testis ACE activity of the same subject were compared in 12 control subjects. We demonstrated that testicular ACE is stable in spermatozoa and the assay of testicular ACE activity is possible. Total testicular ACE activity was approximately twice the membrane testicular ACE activity in all of the subjects tested. The assay of testicular ACE activity in human spermatozoa could be a new method for the assessment of sperm function.