Protein phosphorylation was determined in cultured mouse hepatocytes exposed to an hepatotoxic concentration of acetaminophen (APAP) for selected times up to 12 h. Cultures were radiolabled with 32P-orthophosphoric acid and the cell extracts were analyzed by 2D gel electrophoresis and autoradiography. APAP exposure selectively increased the phosphorylation state of proteins of molecular weight 22, 25, 28, and 59 kDa and decreased the phosphorylation of a 26-kDa protein. Evidence is presented that these changes (1) are dependent on cytochrome P-450 activation of APAP; (2) occur well before enzyme leakage in this in vitro model; (3) are not likely attributed to GSH depletion alone; (4) are in part mimicked by okadaic acid, calyculin A, and cantharidic acid, three structurally distinct inhibitors of protein phosphatases 1 and 2A; and (5) are paralleled by a decline in protein phosphatase activity. The physiological consequences of protein phosphatase inactivation could be significant in APAP overdose since these enzymes are involved in the dephosphorylation of regulatory proteins that control many cell functions. This study also provides the first evidence for disruption in signal transduction pathways as a response to or component of APAP-induced hepatic injury.