Short-lived intracellular proteins, after being marked by multiubiquitination, are degraded by the 26S protease. This large ATP-dependent protease is composed of two multiprotein complexes: the regulatory complex and the 20S proteosome. The selective recognition of ubiquitinated proteins is ensured by the regulatory complex. Using an overlay assay a single 54-kDa multiubiquitin-chain-binding subunit was detected in the regulatory complex of the Drosophila 26S protease. Overlay assay with the recombinant p54 subunit confirmed its ubiquitin-binding property. The recombinant protein showed pronounced preference for higher ubiquitin multimers, in agreement with the known preference of the 26S protease for multiubiquitinated proteins as substrates. To map the ubiquitin-binding domain of the p54 subunit different segments of the recombinant protein were expressed in E. coli and tested by the overlay assay. The p54 subunit carries two independent ubiquitin-binding domains. The central domain carries two highly conserved sequence blocks: the FGVDP sequence (at position 207), which is 100% conserved from yeast till human, and the DPELALALRVSMEE sequence (at position 214), which is 100% conserved in higher eukaryotes with two amino acid changes in yeast. In the C-terminal ubiquitin-binding domain the GVDP sequence motif is repeated and 100% conserved in higher eukaryotes. This domain, however, due to the shorter size of the yeast multiubiquitin-binding subunit, is present only in higher eukaryotes.