We have previously identified a multicatalytic protease (MCP) activity associated with the nuclear scaffold (NS) in hepatocytes and fibroblasts. When we used the chloromethylketone protease inhibitor AAPFcmk, which is targeted to chymotrypsinlike protease activity, we observed a dramatic inhibition of transformation of fibroblasts, with effects that were relatively selective for the NS fraction. Here, we undertook experiments to determine the effects of AAPFcmk on Simian Virus 40-immortalized CWSV1 cells compared with a ros-transformed hepatocyte cell line (NR4) derived from CWSV1. We used biotinAAPFcmk and fluorescent reagents to demonstrate a nuclear chymotrypsinlike protease activity, which is most prominent at the nuclear envelope. The ras-transformed NR4 cells were highly susceptible to growth inhibition in a dose-dependent manner, showing 85% growth inhibition at 50 μmol/L AAPFcmk. In contrast, the immortalized CWSV1 cells were not sensitive at the concentrations (10 to 50 μmol/L) of AAPFcmk tested. In subcellular fractionation studies, the inhibitory effects of AAPFcmk were confined to the NS fraction. The AAPFcmk-induced growth inhibition was accompanied by marked morphological changes in ras-transformed cells, without evidence of overt toxicity. No change in DNA content was observed, but a marked increase in organization of actin cytoskeletal elements was seen. These results suggest that a protease activity associated with the nuclear scaffold has important functions in controlling cytoskeletal filament organization and cell replication. (HEPATOLOGY 1995; 22:1230–1235.).