The YWK-II protein in human spermatozoa is structurally related to the betaA4-amyloid precursor protein of Alzheimer disease and has high similarity with amyloid precusor homologues. Antibodies to the YWK-II protein agglutinate human spermatozoa and may be a potential cause of infertility. In the present study, a yeast two-hybrid system (MATCHMAKER Two-Hybrid System 2; Clontech, Palo Alto, CA) was used to screen a rat ovary cDNA library for potential ligands capable of interacting with the YWK-II component. Mullerian-inhibiting substance was found to interact with the extracellular domain of YWK-II protein. The interaction was confirmed by a binding experiment in vitro and surface plasmon resonance assays. The recombinant Mullerian-inhibiting substance can significantly increase the viability and longevity of human spermatozoa after 5 and 22 h of incubation, presumably through binding the YWK-II component on the sperm membrane. The results of this study indicate that the YWK-II sperm membrane protein may function as a receptor for Mullerian-inhibiting substance.