Riboflavin carrier protein (RCP) is a phosphoglycoprotein (37 kDa) that is well studied in chicken. An immunologically crossreacting protein was identified in mammals and active immunization of male rats and bonnet monkeys with chicken RCP lead to an ,80% reduction in fertility. However, the physiological mechanism responsible for inhibition of male fertility has not been investigated. Moreover, information on the cell type-specific localization and the origin of immunoreactive RCP during spermatogenesis is extremely limited. Hence, studies were carried out to determine the pattern of expression of immunoreactive RCP during spermatogenesis and its role in sperm function in the golden hamster. Immunoreactive RCP was germ cell-specific, found to be associated with the acrosome-organizing region of early spermatids and showed interesting patterns of immunolocalization during late stages of spermiogenesis. Mature spermatozoa exhibited acrosome-specific localization, mainly in the peri-acrosomal membrane. The immunoreactive protein was undetectable in (non)gonadal somatic cells tested. The protein had a molecular mass of 45 -55 kDa and was biosynthesized by round spermatids. The acrosome-specific localization of immunoreactive RCP was unchanged during capacitation, but it was substantially lost during acrosome reaction. Functional studies indicated that treatment of spermatozoa with anti-RCP antibodies did not have any effect on either capacitation or acrosome reaction, but markedly reduced the rate of sperm penetration into zona-free hamster oocytes. These results show the existence of male germ cell-specific immunoreactive RCP, having a potential role in sperm -egg interaction in hamsters. Also the pattern of immunoreactive-RCP localization makes it an ideal marker to monitor development of acrosome in mammalian spermatozoa.