The molecule identified by this antibody has a high molecular mass and is resistant to treatment with SDS/b-mercaptoethanol. The epitope of the TRA 54 antigen probably contains carbohydrate domains as judged from its sensitivity to periodate. Immunohistochemistry revealed that the TRA 54-reactive molecule is located in the supranuclear cytoplasm of caput epididymal epithelial cells and also in luminal sperm. Overall, these results suggest that the molecule recognized by mAb TRA 54 is produced and released by epididymal epithelial cells and subsequently binds to the sperm surface as these cells move down the epididymal duct. The molecules recognized by mAb TRA 54 in epididymal epithelial cells are expressed independently of testicular germ cells and are produced in an androgendependent manner. In this work, we used a combination of column chromatography, SDS-PAGE with in situ digestion and mass spectrometry to identify the protein recognized by mAb TRA 54 in mouse epididymal epithelial cells. Database searches and molecular modeling were used to identify the protein, and fertilization assays in vitro were used to examine the involvement of this protein in egg fertilization. Several molecules secreted into epididymal fluid contribute to the surface modifications involved in sperm maturation. We have previously described an epididymal molecule recognized by mAb TRA 54 that appeared to be released from epididymal epithelial cells and subsequently adhered to luminal sperm. The peculiar expression pattern of this protein suggested that, among other possible functions, this molecule could be involved in sperm-egg recognition or egg penetration. In the present study, we Fingolimod sought to identify the epididymal protein involved in the reactivity with mAb TRA 54 and investigate its possible involvement in fertilization. By using a combination of affinity and anion exchange chromatography, SDS-PAGE with in situ digestion, mass spectrometry and immunodetection we identified a,65 kDa protein that reacted with mAb TRA 54. Curiously, the major mAb TRA 54 immunoreactive band of caput epididymis homogenates had a molecular mass of,260 kDa, which is considerably different from the,65 kDa found for the isolated epididymal protein. This difference in size suggest that the isolated molecule recognized by mAb TRA 54 is part of an SDS/ b-mecaptoethanol-resistant complex of higher molecular mass that may contain more than one type of protein or may be modified by the addition of oligosaccharide chains.