The O-glycosylation sites for equine LHβ (eLHβ) and eCGβ were identified by solid-phase Edman degradation of four glycopeptides derived from the C-terminal region. Both subunits were O-glycosylated at the same 12 positions, rather than the 4–6 sites anticipated. These sites were partially glycosylated, with carbohydrate attachment ranging from 20% to 100% for eCGβ and from 10% to 100% for eLHβ. When the C-terminal peptide containing all but one of the O-linked oligosaccharides was removed by mild acid hydrolysis of either eLHβ or eCGβ, hybrid hormones could be obtained by reassociating eLHα,eFSHα, or eCGα with the truncated β subunit derivatives. These hybrid hormones were identical in LH receptor-binding activity when des(121-149)eLHβ or des(121-149)eCGβ were combined with the same α subunit preparation. Thus, O-glycosylation appears to be responsible for the β subunit contribution to the substantial difference in LH receptor-binding activity between eLH and eCG. Comparison of the equid LH/CGβ sequences with those available for the primate CGβ subunits indicated a greater conservation of glycosylation patterns in the former.