p62 is a scaffolding protein that binds to polyubiquitin. It is involved in the degradation of proteins by the proteasome. To determine if p62 is critical in the development of Mallory bodies (MBs), primary culture hepatocytes from drug-primed mice were studied and the results were compared with normal hepatocytes. Gene-specific gripNA (gp62) was added to the medium of the primary cultures of the hepatocytes to inhibit the expression of p62. Overexpression of p62 was achieved by transfecting the hepatocytes with a plasmid containing green fluorescent protein (GFP) fused p62 (p62-GFP). Gp62 dramatically inhibited MB formation by 94% in drug-primed hepatocytes. The cells transfected with gp62 had decreased protein levels of p62, ubiquitin (Ub), and cytokeratin 8 (CK8). Overexpression of p62 accelerated and enhanced MB formation by 339% in drug-primed hepatocytes. Overexpression of p62 in normal mouse hepatocytes induced MB-like aggresomes that were stained by Ub but not by CK8. The results indicate that p62 is involved in the mechanism of MB formation.