Aging is associated with loss of quality control in protein turnover. The ubiquitin-proteasome pathway is critical to this quality control process as it degrades mutated and damaged proteins. We identified a unique aging-dependent mechanism that contributes to proteasome dysfunction in Drosophila melanogaster. Our studies are the first to show that the major proteasome form in old (43-47 days old) female and male flies is the weakly active 20S core particle, while in younger (1-32 days old) flies highly active 26S proteasomes are preponderant. Old (43-47 days) flies of both genders also exhibit a decline (50%) in ATP levels, which is relevant to 26S proteasomes, as their assembly is ATP-dependent. The steep declines in 26S proteasome and ATP levels were observed at an age (43-47 days) when the flies exhibited a marked drop in locomotor performance, attesting that these are "old age" events. Remarkably, treatment with a proteasome inhibitor increases ubiquitinated protein levels and shortens the life span of old but not young flies. In conclusion, our data reveal a previously unknown mechanism that perturbs proteasome activity in "old-age" female and male Drosophila most likely depriving them of the ability to effectively cope with proteotoxic damages caused by environmental and/or genetic factors.--Vernace, V. A., Arnaud, L., Schmidt-Glenewinkel, T., Figueiredo-Pereira, M. E. Aging perturbs 26S proteasome assembly in Drosophila melanogaster.