We have cloned and sequenced a non-ATPase subunit of the regulatory complex of the Drosophila 26S protease. The gene is present in a single copy in the Drosophila genome. By comparing the nucleotide sequence of the genomic and cDNA clones three exons and two introns were localized. Two transcription start sites were identified 9 bp apart. The deduced protein sequence shows no significant similarity to any other protein in the database. In Drosophila embryos where the 26S protease is present in high concentration, the pool of free subunits of the regulatory complex is very low. Among the free subunits of the regulatory complex the cloned subunit is present in very large excess. This observation raises the possibility that this subunit is in a dynamic equilibrium, exchanging between a free and a particle-bound form, which may have important implications concerning its function.