Müllerian inhibiting substance (MIS), produced by testicular Sertoli cells, is present in adult male serum. The first aim of this study was to determine if MIS is present in seminal plasma. Using an enzyme-linked immunosorbent assay (ELISA), we measured MIS concentrations in seminal plasma from 23 donors exhibiting normal (WHO criteria) sperm qualities, and 169 patients with subnormal sperm parameters. The second aim of this study was to examine a potential relationship between MIS and sperm motility. MIS concentrations in seminal plasma ranged from 0.5 to 3.6 ng/ml in donors and from 0.5 to 17.8 ng/ml in patients. Motility index (MI, mean +/- SEM) for all patient samples was lower compared with donors (113.3 +/- 3.2 and 198.3 +/- 13.5, P < 0.00001), while mean MIS concentration +/- SEM) was higher (4.2 +/- 0.3 and 1.4 +/- 0.2, P < 0.0003). When the patients were stratified into Groups I (motility < 50%, n = 42) and II (motility > 50%, n = 127), the MI (mean +/- SEM) values were 62.3 +/- 3.8 and 130.2 +/- 2.7 respectively (P < 0.0001 for both compared with donors) and mean MIS concentrations (+/- SEM) were 5.4 +/- 0.6 and 3.9 +/- 0.3, respectively (P < 0.0001 and P < 0.001 compared with donors). The inverse relationship between MIS concentration in seminal plasma and motility index suggests that MIS may have a function in modulating motility.