The sea urchin egg vitelline envelope (VE) is composed of eight major glycopolypeptides that are heavily mannosylated and contain fucose and N-acetylglucosamine moieties based on lectin staining. In the present study, the macromolecular composition of the VE and the potential role of a purified VE glycoprotein in initial gamete binding was investigated. The VE components were solubilized from the surface of intact, dejellied eggs with dithiothreitol in divalent cation-free seawater, and analyzed using native, reduced electrophoresis and immunoblotting. Three major VE glycoproteins, VE-A, VE-B and VE-C, and one minor component, VE-D, were identified with antisera against whole VE preparations and against glutaraldehyde-fixed, unfertilized eggs. The electrophoretically purified glycoproteins resolved into a common subunit doublet and one unique subunit each of decreasing size on blots of sodium dodecylsulfate polyacrylamide gels. Lectin affinity chromatography was used for analysis and purification of reduced VE components; a glycoprotein eluted from Con A columns with methyl-mannoside comigrated with VE-B when analyzed by immunoblotting. Whole VE preparations and VE-B obtained from Con A columns were found to inhibit fertilization when preincubated with sperm, thus directly establishing a role for VE-B in gamete binding.