Inhibin B is bidirectionally secreted by Sertoli cells, basal secretion into the circulation exerts negative feedback on follicle-stimulating hormone secretion, and serum inhibin B is considered a marker of spermatogenesis. The precise role of apical secretion is unknown. The objective of our work was to study the relationship between seminal inhibin B and spermatogenesis. Dimeric inhibin B was measured by immunoassay in seminal plasma of volunteers with normozoospermia (n = 10, group 1), in men after vasectomy (n = 10, group 2), and in men with azoospermia (n = 50, group 3). Testicular biopsy and testicular sperm extraction were performed in men with azoospermia. Seminal inhibin B levels were higher in men in group 1 than in men in groups 2 and 3 (P <.0001). In seminal plasma, inhibin B presents a positive correlation with alpha glucosidase activity (r =.37, P =.002). Seminal inhibin B is inversely related with serum FSH (r = -.58, P <.001), and presents a weak positive correlation with serum testosterone concentration (r =.29, P =.03). No difference was found between inhibin B levels in seminal plasma of patients with nonobstructive or obstructive azoospermia, and between positive or negative outcome of TESE. We conclude that inhibin B secretion by Sertoli cells is differentially regulated. The contribution of accessory sex glands limits the use of seminal plasma inhibin B as a marker of spermatogenesis.