The biochemical and ultrastructural changes in the envelope of the Xenopus laevis egg that occur during oviposition and fertilization have been thoroughly studied (Hedrick, J. L., and Nishihara, D. M., Methods Cell Biol. 36, 231-247, 1991; Larabell, C. A., and Chandler, D. E., J. Electron Microsc. Tech. 17, 294-318, 1991). However, the biological significance of these changes with respect to gamete interaction has been unclear. In the current study, it was found that changes in the envelope are directly responsible for regulating sperm-egg adhesion, an initial step of fertilization. As a result of these transformations, sperm bind only to unfertilized oviposited eggs, not to oocytes or coelomic eggs. In addition, they do not bind to fertilized eggs. The molecular and cellular basis of the regulation of the sperm binding process was investigated in the context of our recent findings that two structurally related envelope glycoproteins, gp69/64, serve as sperm receptors during fertilization (Tian, J.-D., Gong, H., Thomsen, G. H., and Lennarz, W. J., J. Cell Biol. 136, 1099-1108, 1997). Although the purified gp69/64 glycoproteins isolated from the oocyte or coelomic egg envelopes exhibited sperm binding activity, when these proteins are part of the intact oocyte or coelomic egg envelopes, they are not accessible to either anti-gp69/64 antibodies or to sperm. During the conversion from the coelomic to the vitelline envelope, the gp69/64 sperm receptors become exposed on the surface, an event that correlates with proteolytic cleavage of gp43 and accompanying ultrastructural alterations in the envelope. Conversely, after fertilization, when the vitelline envelope of the egg is converted to the fertilization envelope of the zygote, limited proteolytic cleavage of the sperm receptor results in loss of sperm binding activity. In addition, formation of a fertilization layer on top of the structurally altered VE adds another physical block to sperm binding. These results provide new insights into structure-function relationships between envelope components of the anuran egg, and provide further evidence supporting the key role of gp69/64 as sperm receptors during X. laevis fertilization.