In the present report, valosin-containing protein (VCP) was present in Mallory bodies (MBs). To determine if VCP plays a role in MB formation, primary cultured hepatocytes from drug-primed mice that spontaneously form MBs in vitro were studied. The results were compared with control normal hepatocytes. Gene-specific FITC-labeled gripNA (gVCP) was added to the medium of the primary cultures to inhibit the expression of VCP. gVCP increased MB formation by 230% in drug-primed mouse hepatocytes compared with primed liver cells where no VCP oligos were added. Blocking VCP expression induced both multiple small ubiquitin (Ub) and cytokeratin (CK) aggregates to form within the cytoplasm in normal mouse hepatocytes. Inhibition of VCP expression in both drug-primed and control hepatocytes caused a decrease in proteasome chymotrypsin-like (ChT-L) activity. Overexpression of VCP was achieved by transfecting the hepatocytes with a plasmid containing green fluorescent protein (GFP)-fused VCP (pVCP-GFP). Overexpressed VCP was located in both the cytoplasm and nucleus of pVCP-GFP overexpressing drug-primed hepatocytes. VCP was also concentrated within MBs. MB formation was not decreased by the overexpression of VCP in the cells. These results indicate that VCP plays an important role in inducing MB formation, probably through its molecular chaperone function in the ubiquitin-proteasome system (UPS).