We updated the two-dimensional protein database for mouse liver. Microsomal and cytosolic fractions of the liver proteins from male mice were separated by two-dimensional electrophoresis. The proteins were identified by Matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS) on the basis of peptide mass fingerprinting, following in-gel digestion with trypsin and matching with the theoretical peptide masses of all known proteins from all species. Approximately 5800 spots, excised from 14 two-dimensional gels, were analyzed which resulted in the identification of about 2500 proteins that were the products of 328 different genes. The database includes 112 newly identified gene products. The fractionation prior to two-dimensional electrophoresis was essential for the detection of the new proteins, 55% of which were found in the microsomal and 35% in the cytosolic fraction. The more frequently identified proteins in the various gels were heat shock proteins, house-keeping enzymes, such as ATP synthase chains, disulfide isomerase, and structural proteins, such as tropomyosin. About 45% of the identified proteins were detected 1-3 times, 45% 4-9 times, and the rest 10 or more times. Most proteins were represented by many spots. In average, about 18-20 spots were detected per gene product.