Leydig cells are located in the interstitium of the testis and function as the primary site for testosterone biosynthesis. Leydig cell development and steroidogenic function are dependent upon pituitary-derived LH. Circulating LH levels in prepubertal mammals are low but rise sharply during puberty, inducing terminal differentiation of immature Leydig cells into adult Leydig cells. The molecular mechanisms involved in LH action on differentiation specific gene expression and initiation of steroidogenic function in immature Leydig cells are poorly understood. Members of the CCAAT/enhancer-binding protein (C/EBP) family of basic region/leucine zipper transcription factors have previously been implicated as regulators of terminal differentiation in several cell types. In the present study we have investigated the possible involvement of C/EBP proteins in regulating LH-dependent gene expression in Leydig cells. We have detected the expression of one family member, C/EBPbeta, in Leydig cells. C/EBPbeta messenger RNA and protein levels were significantly higher in mature adult Leydig cells than in immature cells, displaying an expression pattern similar to those of other developmentally regulated genes in Leydig cells such as steroidogenesis acute regulatory (StAR) protein and 3beta-hydroxysteroid dehydrogenase. C/EBPbeta messenger RNA and protein levels also increased when immature Leydig cells were treated with either hCG, a functional analog of LH (hCG/LH), or (Bu)2cAMP. To confirm that hCG/LH and (Bu)2cAMP were acting specifically on Leydig cells, we studied their effects on C/EBPbeta expression in an established Leydig cell line (MA-10). hCG and (Bu)2cAMP treatment also induced the expression of C/EBPbeta and StAR in MA-10 cells, coincident with stimulation of steroid production in these cells. (Bu)2cAMP treatment did not alter the subcellular localization of C/EBPbeta protein in MA-10 cells, suggesting that the increase is due to stimulation of C/EBPbeta expression. We conclude that expression of C/EBPbeta is regulated by hCG/LH in Leydig cells and that C/EBPbeta may play a significant role in LH-regulated Leydig cell differentiation and function.