In spermatogenesis, Sertoli cells serve as supporting cells for the proliferation and differentiation of germ cells. However, it appears that Sertoli cell function is regulated by adjacent spermatogonial cells in the testis, since expression of lipocalin-2 mRNA, which encodes an iron-siderophore-binding protein, is barely detectable in Sertoli cells of germ cell-deficient W/W^v mice, and more abundantly expressed in jsd/jsd mice. By employing a co-culture system comprising immortalized Sertoli cells (designated as Sertoli-B) and c-Kit⁺ spermatogonial cells from 7-day-old mouse testis, we found that lipocalin-2 gene transcription in Sertoli cells is induced by a factor secreted from spermatogonial cells. Transfection of Sertoli-B cells with a series of reporter constructs encompassing an upstream region of the mouse lipocalin-2 gene revealed that an NF-B binding consensus sequence in the proximal region of lipocalin-2 gene is responsible for transcriptional activation. A major NF-B component, p65, bound to this region and translocated from the cytoplasm to the nucleus upon stimulation with spermatogonial cell conditioned medium. Moreover, short interference RNA directed to p65 or a dominant-negative form of IB suppressed the spermatogonial cell factor-mediated transcription of lipocalin-2. However, NF-B-activating inflammatory molecules, such as interleukin-1 and lipopolysaccharide, did not induce lipocalin-2 mRNA in Sertoli-B cells and the expression of lipocalin-2 was unaffected in the testis of IkB-deficient mice. These results demonstrate that spermatogonial cells regulate lipocalin-2 gene expression in Sertoli cells in a manner distinct from that employed by immune cells.