Our recent Sertoli cell (SC) studies showed that the cJUN NH2-terminal kinase (JNK) and inducible cyclooxygenase-2 (COX-2) pathways are key regulatory components of interleukin (IL; IL-1, IL-1, and IL-6) expression and START-domain containing StARD1 and StARD5 proteins. IL-1 regulates SC autocrine/paracrine activities and subsequently influences developing germ cells and spermatogenesis. This study was designed to evaluate whether IL-1 mediates high-output inducible nitric oxide synthase (iNOS) expression and nitric oxide (NO) production in these specialized epithelial cells and characterize gonadotropin and cytokine-regulation of NO. Purified SC were maintained in serum-free cultures and treated with FSH (100 ng-1 µg/ml) or IL-1 (10 ng/ml) in time-course studies. To determine obligatory intracellular pathways, treatments were conducted with or without activity inhibitors: COX-2 selective (NS-398, 10 µM) or JNK (SP600125, 10 µM) for 1, 3, 6, and 24 h. NOS mRNAs and proteins were evaluated by RT-PCR and Western analysis, respectively. NO and reactive oxygen species (ROS) were measured by flow cytometry and ELISA. IL-1 transiently induces intracellular NO (30-min) but not reactive oxygen species (ROS). Subsequently, iNOS mRNA and protein expression (3-6 h) significantly increased following IL-1 but not FSH stimulation, and in time-dependent manner, markedly increased extracellular NO (24 h, 8-fold). No change in the constitutive eNOS isoform was observed. Inhibition of JNK, but not COX-2, activity inhibits IL-1-induced iNOS expression and NO production. Such findings suggest that intra and extracellular NO within the tubule may alert Sertoli cells monitoring the microenvironment to an aberrant cytokine, triggering anti-oxidant and anti-inflammatory activities to avoid disruption of spermatogenesis.