The number of adult Leydig cells is one of the factors controlling testosterone secretion by sexually mature testis, and it depends on the proliferative capacity of prepubertal Leydig cells. We investigated here whether this capacity is controlled by leptin because this hormone regulates proliferation in other cell types and has a crucial role in male fertility. Our data show that prebupertal Leydig cells express the Ob/Rb form of leptin receptor and are thus direct targets of this hormone. The analysis of G1/S-phase cyclins by quantitative (real-time) RT-PCR and Western blot points to the leptin-induced decrease in cyclin A2 and subsequent increase in cyclin D1 expression that precedes a leptin-triggered decrease in the number of prepubertal Leydig cells. Quantitative assessments of DNA synthesis by bromodeoxyuridine incorporation and of cycling cell population by Ki67 immunocytochemistry indicate that leptin decreases the cell number by inhibiting cell division and increases mRNA levels of Leydig cell differentiation markers such as relaxin-like factor. Immunohistochemistry of cyclin D1 and relaxin-like factor pointed to the parallel increase of their expression coinciding with the onset of Leydig cell differentiation. Moreover, leptin-treated Leydig cells display increased expression of another differentiation marker (3beta-hydroxysteroid dehydrogenase) that is abolished by knocking down cyclin D1 with small interference RNA. Altogether, our data show that leptin inhibits division of prepubertal Leydig cells via a cyclin D-independent mechanism and suggest that cyclin D1 might be involved in leptin-induced differentiation of Leydig cells.