Luteinizing hormone (LH) and human chorionic gonadotropin (hCG) are two glycoprotein hormones regulating development and reproduction. Despite binding the same receptor (LHCGR), they elicit different intracellular signaling. In vitro non-equivalence of hCG and LH was previously demonstrated in human primary granulosa cells. The aim of this study is to compare the effects of LH and hCG in mouse primary Leydig cells in vitro, naturally expressing the murine receptor (l hr). Although hCG is absent in mice, lhr retains the ability to binds both these human gonadotropins and shares 80% identity with the human LHCGR. Testis from 3/5-months-old C57BL6 mice were collected and Leydig cells were isolated by density gradient, plated and treated by increasing doses of recombinant LH and hCG (1 pM100 nM range). We evaluated cAMP production and testosterone synthesis by ELISA, ERK1/2 and CREB phosphorylation by Western blotting, gene expression by real-time PCR. Despite no different gene expression and testosterone production was found, hCG is about 10-fold more potent than LH in inducing cAMP accumulation (hCG EC50=18.64±10.14 pM; LH EC50=192.00±53.96 pM; MannWhitneys U-test; P<0.05; n=4) and higher levels of ERK1/2 (hCG: 6646.1±1492.1; LH: 2214.1±464.8 relative units) and CREB (hCG: 2318.3±495.9; LH: 1360±395.3 relative units) (means±S.E.M.) (Two-way Anova and Bonferroni post-test, P<0.05; n=4) phosphorylation at the highest dose used (100 nM). We demonstrated that hCG and LH treatment results in quantitatively but not qualitatively different signaling in Leydig cells in vitro, differing to that previously described in human primary granulosa cells naturally expressing the human receptor. The high levels of cAMP/PKA and pERK1/2 activation induced by hCG may rely on not complete aminoacids identity (about 75%) between the LHCGR and lhr hinge regions, which are fundamental for proper hormone binding and signal transduction. Our results support that LH and hCG are not equivalent at molecular levels.