In the present study we have established an immunochemical mapping of equine Chorionic Gonadotropin (eCG/PMSG) using three monoclonal antibodies (mAbs), namely the antibodies ECG01, E10 and D7, raised against the native hormone. These antibodies do not bind to reduced, alkylated hormone, suggesting that they recognize discontinuous rather than continuous epitopes. We have also assessed the reactivity of mAbs towards human CG, and ovine, porcine, equine and bovine LH and FSH. The antigenic determinant recognized by ECG01 is localized on the alpha-subunit of equine gonadotropins and of human CG and LH. The epitopes recognized by E10 and D7 mAbs appear to be very similar and are present on the beta-subunit of eCG and of LHs from all species tested, except hLH, as well as on porcine and equine FSHs. Attempts to specify the amino-acid residues involved in these epitopes suggest that ECG01 mAb might preferentially bind to residues around position 70 whereas the region around disulfide bridges Cys-88-Cys-90 might be involved in the epitopes recognized by D7 and E10 mAbs. Topographical relationships of epitopes show that ECG01 mAb never binds to eCG simultaneously with either D7 or E10 mAbs. Furthermore, simultaneous binding of D7 and E10 mAbs on eCG could not be achieved. Thus, these three epitopes appear to be closely located on the surface of eCG. Finally, ECG01 mAb inhibits eCG binding to LH and FSH receptors, suggesting that its antigenic site is closely related to hormone-receptor interaction site(s).