Nitric oxide (NO) has emerged as an intracellular and intercellular messenger in a number of biological systems. In the present study, we demonstrated that NO was produced by cultured rat Leydig cells, and that inducible nitric oxide synthase (iNOS) messenger RNA (mRNA) was expressed in Leydig cells. NO was measured as nitrite with the method of Griess. Although unstimulated Leydig cells produce little NO, interleukin-1 beta (IL-1 beta) markedly increased NO production. NO production was inhibited by the NOS inhibitor, NG-monomethyl-L-arginine. Northern blot analysis showed that iNOS mRNA was little expressed in freshly isolated immature Leydig cells, but that iNOS mRNA levels were increased by the addition of IL-1 beta in a dose-dependent manner at the concentration up to 10 ng/ml. The levels of iNOS mRNA were increased as early as 3 h after the addition of IL-1 beta and persisted for up to 24 h. In adult Leydig cells, IL-1 beta stimulated iNOS mRNA expression. Immunocytochemical analysis demonstrated iNOS-like immunoreactivity in the cytoplasm of Leydig cells. These results indicate that NO is produced in Leydig cells and suggest that NO might be involved in the physiological function of Leydig cells.