Nucleoside diphosphate (NDP) kinases, responsible for the synthesis of nucleoside triphosphates and produced by the nm23 genes, are involved in numerous regulatory processes associated with proliferation, development, and differentiation. Their possible role in providing the GTP/ATP required for sperm function is unknown. Testis biopsies and ejaculated sperm were examined by immunohistochemical and immunofluorescence microscopy using specific antibodies raised against Nm23-H5, specifically expressed in testis germinal cells and the ubiquitous NDP kinases A to D. Nm23-H5 was present in sperm extract, together with the ubiquitous A and B NDP kinases (but not the C and D isoforms) as shown by Western blotting. Nm23-H5 was located in the flagella of spermatids and spermatozoa, adjacent to the central pair and outer doublets of axonemal microtubules. High levels of NDP kinases A and B were observed at specific locations in postmeiotic germinal cells. NDP kinase A was transiently located in round spermatid nuclei and became asymmetrically distributed in the cytoplasm at the nuclear basal pole of elongating spermatids. The distribution of NDP kinase B was reminiscent of the microtubular structure of the manchette. In ejaculated spermatozoa, the proteins presented specific locations in the head and flagella. Nm23/NDP kinase isoforms may have specific functions in the phosphotransfer network involved in spermiogenesis and flagellar movement.