Loss of the endoplasmic reticulum resident chaperone calmegin leads to the production of sterile sperm that do not bind to the egg zona pellucida (M. Ikawa et al., 1997, Nature 387, 607–611). In the present study, we demonstrate that calmegin −/− sperm were defective in migrating into the oviducts and in binding to the egg plasma membrane. Despite the impaired adhesive function, calmegin −/− sperm could fertilize eggs when zonae pellucidae were partially dissected, and eggs fertilized in this manner could develop normally to term. Since these sperm characteristics were similar to those found in fertilin β −/− sperm, we investigated the interaction of calmegin with fertilin β. Using immunoprecipitation techniques, calmegin was found to bind to sperm membrane proteins, fertilin α and β, during spermatogenesis. The binding was specific to calmegin: another endoplasmic reticulum chaperone calnexin, a calmegin homologue, was not able to bind to fertilin α and β. In the calmegin −/− mice, a loss of heterodimerization of fertilin α and β was observed and fertilin β was not detectable in mature sperm. The data not only explain why the calmegin and fertilin β knockout mouse lines share a common infertile phenotype, but also reveal the importance of the maturation of sperm membrane proteins in the endoplasmic reticulum.