To determine intracellular factors influencing the sensitivity of cancer cells to tumor necrosis factor-alpha (TNF-alpha), we studied the expression of intracellular proteins in TNF-alpha-resistant cKDH-8/11 and -sensitive KDH-8NK rat hepatoma cell lines using the technique of two-dimensional gel electrophoresis (2-DE). From the 2-DE patterns, ii was demonstrated that TNF-cr-resistant cKDH-8/11 cells had increased levels of protein of molecular weight (M-r,) 22 500 and isoelectric point (pl) 5.2, compared with TNF-alpha-sensitive KDH-8/YK cells. Therefore, we excised cyanogen bromide (CNBr) fragments of proteins in the spot for N-terminal sequencing. Microsequencing for the CNBr fragments identified the protein as rat phosphatidylethanolamine-binding protein. These findings suggest that the intracellular phosphatidylethanolamine-binding protein could be one of the factors responsible for the resistance of cKDH-8/11 cells to TNF-alpha-induced cell death.