Abstract The 26S proteasome is a multicatalytic protease responsible for regulated intracellular protein degradation. Its function is mediated by three main catalytic activities: (a) chymotrypsin-like (CT-L), (b) trypsin-like, and (c) peptidylglutamyl peptide hydrolysing (PGPH). Proteasome inhibition is an emerging therapy for many cancers and is a novel treatment for multiple myeloma. Here, we profile the contributions of the three catalytic activities in multiple myeloma cell lines and compare the specificity and cytotoxicity of the novel proteasome inhibitor BzLLLCOCHO and inhibitors PS-341 (Velcade, bortezomib) and MG-132. Using fluorogenic substrates and an active site-directed probe specific for proteasome catalytic subunits, we show differential subunit specificity for each of the inhibitors. Addition of BzLLLCOCHO strongly inhibited all three catalytic activities, treatment with PS-341 completely inhibited CT-L and PGPH activities, and treatment with MG-132 resulted in weak inhibition of the CT-L and PGPH activities. Multiple myeloma cells were more sensitive to induction of apoptosis by PS-341 and MG-132 than BzLLLCOCHO. This study emphasizes the need for further investigation of the effects of these compounds on gene and protein expression in the cell to allow for the development of more specific and targeted inhibitors. (Cancer Res 2006; 66(12): 6379-86)