Three equine CG (eCG) forms with identical amino acid sequences but different mol wt and monosaccharide compositions were isolated from a crude eCG preparation and designated eCG-L (low mol wt), eCG-M (medium mol wt), and eCG-H (high mol wt). No differences in primary structure between each form and the known sequence of eCG were observed. SDS-PAGE of these preparations under reducing conditions revealed that the mol wt differences between them were due only to the different sizes of their beta-subunits. Carbohydrate compositions suggested an increase in O-glycosylation in the higher mol wt forms. N-Linked glycopeptide fragments obtained from eCG beta-subunits by endoproteinase Lys-C digestion had identical electrophoretic mobilities. Thus, the different molecular sizes of the beta-subunits were associated only with disparities in O-glycosylation of their C-terminal extension. When tested in a LH and several FSH radioligand assay systems, eCG-H proved to have significantly lower receptor-binding activities than eCG-L and eCG-M. Endo-beta-galactosidase digestion increased the FSH receptor-binding activity of all eCG forms; however, partially deglycosylated eCG-H remained the least active form. Thus, the O-linked oligosaccharides of eCG-H exert a negative influence on its receptor-binding activity.