We investigated the expression and activity of mitogen-activated protein kinase (MAPK) in human hepatocellular carcinoma (HCC). MAPK expression was determined in five human tumors and five normal tissues (adjacent non-neoplastic liver) by Western blotting using specific antisera raised against four MAPK pathway intermediates: Erk-1, Erk-2 (extracellular-signal regulated kinases), Mek-1 and Mek-2 (mitogen activated protein kinase kinases). There was a significant increase in Erk-1, Erk-2, Mek-1 and Mek-2 expression in particulate and cytosolic fractions prepared from tumor specimens as compared with the adjacent normal control tissues. The functional activity of both membrane and cytosolic Erk-2, determined by phosphorylation of myelin basic protein (MBP), was significantly increased in tumor specimens as compared to normal (membrane: 321%+/-50%, p<0.05; and cytosol: 597%+/-233%, p<0.05 percent of normal tissue). These data demonstrate for the first time a significant increase in MAPK expression and functional activity in human HCC. Because of the important role that the MAPK pathway plays in cellular growth and differentiation, overexpression of MAPK may be of critical importance to the formation and maintenance of human hepatocellular carcinoma.