Lutropin (LH), follitropin (FSH), thyrotropin (TSH) and choriogonadotropin (CG) are assembled of two non-covalently alpha (α) and beta (β) subunits. We studied the discontinuous antigenic regions recognized by a monoclonal anti-hLH antibody designated as LH05 which binds to hLH, hCG and hTSH and does not cross-react with either the free subunits or hFSH. LH05 and an antibody designated HT13, recognizing an epitope partly comprizing the α64–76 region, did not bind simultaneously to hCG. Furthermore, LH05 was unable to combine with an anti-peptide antibody (LHP03) directed to residues 43–52 of hLHβ. Thus, LH05 recognizes an epitope partly overlapping with those recognized by HT13 and LHP03. Using various hybrid molecules, we showed that the human α-subunit plays a critical role in the assembly of the epitope that, in contrast, contains amino acid residues conserved in the various β-subunit of several species. Together, our results suggest that the amino acids Leu49-Pro50, Tyr59-Arg60 and Leu86-Ser87 in the hLHβ and the α64–76 region are probably included in the epitope recognizedby LH05 which appeared to be not accessible on the CG/LH receptor.