Subcellular fractionation increases the probability of detection of low-abundance proteins. We prepared a fraction highly enriched in mitochondrial proteins from rat liver. The proteins were analyzed by two-dimensional (2-D) electrophoresis using broad and narrow-range immobilized pH gradient strips, and identified by matrix assisted laser desorption/ionization-mass spectrometry (MALDI-MS). 192 different gene products were detected, of which approximately 70% were enzymes with a broad spectrum of catalytic activities. Most of the identified proteins were detected in other rat protein samples as well, which were analyzed in our laboratory. Eight gene products were detected for the first time. These were represented by one spot each, whereas most of the frequently detected proteins were represented by multiple spots. In average, approximately 10-15 spots corresponded to one gene product.