Neutral endopeptidase (NEP, metallo-endopeptidase EC 3.4.24.11; enkephalinase, neprilysin, CD10, CALLA) represents a major regulator of bioactivity of natriuretic peptides. C-type natriuretic peptide (CNP) is present in high levels in epididymis and seminal plasma. However, detailed expression pattern and CNP-related function of NEP in the epididymis are unknown. Comparison of NEP protein levels in various organs revealed an extremely high expression in human and mouse epididymis. NEP was localized exclusively to apical (luminal) parts of epithelial cells. In man, strong NEP-immunoreactivity was associated with epithelia of efferent ducts and the epididymal duct including stereocilia. Segment-by-segment analysis in mouse revealed a distinct distribution along the epididymal duct. We also found the CNP receptor guanylyl cyclase B (GC-B) in epithelial cells of the epididymal duct. Two different NEP inhibitors decreased CNP degradation and increased CNP/GC-B-induced cGMP production by epididymal membranes, suggesting a functional involvement of NEP. Data indicate an important, previously neglected, role of NEP for regulation of luminal factors in the epididymis and suggest a novel role for CNP/GC-B in the epididymal epithelium, presumably in context of local water balance.