A sulfoglycolipid immobilizing protein, termed SLIP1, is a conserved germ cell membrane protein that has been shown in vitro to bind specifically to the mammalian germ-cell-specific sulfoglycolipid, sulfogalactosylglycerolipid (SGG). SLIP1 was extracted from the mouse sperm surface by a sucrose/ATP/EDTA solution. Indirect immunofluorescence and immunoprotein A-gold labeling of live mouse sperm indicate that SLIP1 was present on the acrosomal plasma membrane. Inclusion of low concentrations of exogenous purified SLIP1 in the in vitro mouse sperm-egg binding assay culture significantly decreased the number of sperm bound per egg. Inhibition was heat labile. SLIP1 on sperm, rather than that on eggs, appeared to be important for the binding process since preexposure of sperm to anti-SLIP1 decreased sperm binding to the eggs. Eggs preincubated with anti-SLIP1 were unaffected. Immunoblotting studies confirmed that SLIP1 was not present on the zona pellucida (ZP). SLIP1 binding ligand(s) on eggs were also involved in sperm-ZP binding since preincubation of eggs with exogenous SLIP1 before gamete coincubation significantly reduced the number of sperm bound per egg. The results suggest that SLIP1 is involved in mouse sperm-ZP binding.