A precise packaging of the paternal genome during spermiogenesis is essential for fertilization and embryogenesis. Most of the nucleosomal DNA supercoiling must be eliminated in elongating spermatids (ES), and transient DNA strand breaks are observed that facilitate the process. Topoisomerases have been considered as ideal candidates for the removal of DNA supercoiling, but their catalytic activity, in the context of such a major chromatin remodeling, entails genetic risks. Using immunofluorescence, we confirmed that topoisomerase II beta (TOP2B) is the type II topoisomerase present in ES between steps 9 and 13. Interestingly, the detection of TOP2B was found coincident with detection of tyrosyl-DNA phosphodiesterase 1 (TDP1), an enzyme known to resolve topoisomerase-mediated DNA damage. The presence of gamma-H2AX (also known as H2AFX) coincident with DNA strand breakage was also confirmed at these steps and indicates that a DNA damage response is triggered. Active DNA repair in ES was demonstrated using a fluorescent in situ DNA polymerase activity assay on squash preparations of staged tubules. In the context of haploid spermatids, any unresolved double-strand breaks, resulting from a failure in the rejoining process of TOP2B, must likely rely on the error-prone nonhomologous end joining, because homologous recombination cannot proceed in the absence of a sister chromatid. Because this process is part of the normal developmental program of the spermatids, dramatic consequences for the genomic integrity of the developing male gamete may arise should any alteration in the process occur.