Most regulated proteolysis in eukaryotes is carried out by the 26S proteasome. This large, multisubunit complex comprises a catalytic core particle (20S proteasome) and a regulatory particle (19S regulator) capping each end. In Drosophila, about a third of the 32 proteasome subunits are found to have testis-specific isoforms, encoded by paralogous genes. Here,we characterize in detail the spermatogenic expression of the core particle subunit Prosα6 (Pros35) and its testis-specific isoform Prosα6T. Using GFP-tagged transgenes, it is shown that whereas the Prosα6 subunit is expressed in early stages of spermatogenesis, gradually fading away following meiosis, the testis-specific Prosα6T becomes prominent in spermatid nuclei and cytoplasm after meiosis, and persists in mature sperm. In addition, these subunits are found in numerous `speckles' near individualization complexes, similar to the previously described expression pattern of the caspase Dronc (Nedd2-like caspase), suggesting a link to the apoptosis pathway. We also studied the phenotypes of a loss-of-function mutant of Prosα6T generated by targeted homologous recombination. Homozygous males are sterile and show spermatogenic defects in sperm individualization and nuclear maturation, consistent with the expression pattern of Prosα6T. The results demonstrate a functional role of testis-specific proteasomes during Drosophila spermatogenesis.