Sulfogalactosylglycerolipid (SGG) is the major sulfoglycolipid of mammalian male germ cells. Like other sulfoglycolipids, SGG is believed to be involved in cell-cell/extracellular matrix adhesion. Specifically, we investigated whether sperm SGG played a role in sperm-egg interaction. Initially, we produced an affinity-purified, rabbit polyclonal immunoglobulin (Ig) G antibody that specifically recognized SGG (anti-SGG). Indirect immunofluorescence using anti-SGG IgG localized SGG to the convex and concave ridges and the postacrosome of the mouse sperm head. Pretreatment of sperm with anti-SGG IgG/Fab inhibited sperm-zona pellucida (ZP) binding in vitro in a concentration-dependent manner (to a maximum of 62%). This inhibition was observed at the level of primary binding. Sperm treated with anti-SGG IgG underwent the spontaneous and ZP-induced acrosome reaction at the same rate as control sperm treated with preimmune rabbit serum IgG. Fluorescently labeled SGG liposomes were shown to associate specifically with the egg ZP, whereas fluorescently labeled liposomes of galactosylglycerolipid (SGG's parental lipid) and phosphatidylserine (negatively charged like SGG) did not. Furthermore, coincubation of SGG liposomes with sperm and isolated ZP inhibited sperm-ZP binding in a concentration-dependent manner. These results strongly suggest an involvement of sperm SGG in direct binding to the ZP.