A transgenic (TG) mouse model carrying a 2-kb murine LH receptor (LHR) promoter/β-galactosidase (β-GAL) fusion gene was created to study the regulatory function of the 5′-flanking region of the murine LHR gene. Of the five TG mouse lines produced, three displayed high β-GAL expression in the testis, but none showed any expression in the ovary. In addition, all mouse lines of both sexes expressed β-GAL consistently in the brain, most prominently in hippocampus, hypothalamus, midbrain, and cortex. Weak staining was found in a few pituitary samples. All other tissues examined were negative for transgene expression. In support of sex-specific gonadal expression of the transgene, transient transfection of the LHR/β-GAL gene construct into immortalized mouse granulosa (KK-1) and Leydig (mLTC-1) tumor cells revealed a more than 5-fold higher expression level in the Leydig cells. Histological examination of the TG testes demonstrated strongβ -GAL expression in Leydig cells, but, unexpectedly, also in elongating spermatids of adult age and in some spermatogonia of the neonatal testis. The functional significance of the latter findings remains open. The transgene was only expressed in adult Leydig cells; no expression was found in the fetal population of these cells. Hence, these findings indicate that the immediate 2-kb fragment of the murine LHR 5′-flanking sequence is transcriptionally active only in adult Leydig cells and certain brain areas, but other promoter sequences are apparently needed for ovarian and fetal testicular expression of the LHR gene.