A monoclonal antibody (6964M) was generated against the envelope component gp69/64 of Xenopus laevis eggs. On indirect immunofluorescence using this antibody, the positive reaction was seen on the surface of both vitelline envelope (VE) and coelomic envelope (CE). On immunoelectron microscopy, gp69/64 was preferentially distributed on the thick bundles forming the edge of the tunnel openings on CE, and this distribution pattern was fundamentally inherited by VE. Counting the number of immunogold particles indicated that VE has about twice as many particles as CE, with a 3-4 times higher density at the animal pole than vegetal pole. The number of sperm bound to CE was small, being approximately one-twentieth of the number of sperm bound to VE. An extremely small number of sperm (< 2 per animal hemisphere) was found to bind to VE* of activated eggs as a background. The sperm binding to CE was inhibited by pretreatment of the envelopes with 6964M or in the presence of purified gp69/64 from VE on insemination, confirming that sperm binding is mediated by gp69/64 exposed on the CE surface. In spite of at most a 2-fold increase in the amount of exposed gp69/64, the sperm binding increased about 20-fold upon CE-to-VE conversion, suggesting that the increase in the amount of exposed gp69/64 is itself insufficient to explain the increase in the number of bound sperm.