Objective: To investigate the expression and cellular distribution of regulated upon activation normal T-cell expressed and secreted (RANTES) in the male reproductive system. Design: Basic research. Setting: University academic medical center. Patient(s): Three adult male organ donors. Intervention(s): None. Main Outcome Measure(s): Reverse transcriptase-polymerase chain reaction (RT-PCR), in situ hybridization, immunohistochemical staining, and immunofluorescence staining were used to examine the distribution of RANTES in human and mouse epididymis. Western blot was used to quantitate the levels of RANTES expression in mouse epididymis on postnatal days. Immunofluorescence staining was applied to detect RANTES association with spermatozoa from mouse epididymis. Result(s): The location of RANTES was restricted to ciliated cells of the efferent duct and apical, narrow, and basal cells of the epididymal ducts, in both humans and mouse. RANTES-positive basal cells were only identified in the epididymal ducts in humans. The signals of RANTES were first detected on day 28 and increased during mouse sexual maturation. We also observed that RANTES was bound on both normal and defective epididymal sperm, but in different patterns. Conclusion(s): RANTES is constitutively expressed in the epididymis and secreted into the lumen of epididymis throughout sexual maturity, and differentially associates with viable and defective spermatozoa. © 2010 American Society for Reproductive Medicine.