Mitochondrial proteins exert important functions in biological pathways, particularly they are involved in apoptotic processes. We applied proteomics technologies to analyze the mitochondrial proteins of the neuroblastoma cell line IMR-32, which is often used in apoptosis studies. The proteins were analyzed by two-dimensional (2-D) electrophoresis followed by matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS). 185 different gene products were identified, of which approximately 55% were enzymes with a broad spectrum of catalytic activities. Sixteen proteins were detected only in this preparation, the others have been detected in two or more protein samples analyzed by MS in our laboratory, The 16 unique gene products were represented by one spot each, whereas most of the frequently detected proteins were represented by multiple spots. In average, approximately 5-10 spots corresponded to one gene product. For two thirds of the proteins identified, an annotation exists in the SWISS-PROT database about their subcellular location. They are mainly described as mitochondrial, 8 as endoplasmic reticulum, 3 as peroxisomal and only 12 low-abundance proteins are described as cytosolic proteins. The list includes about 30 unknown, hypothetical or poorly described gene products. Some of them are represented by strong spots and the present study shows that they are indeed expressed and are localized in the mitochondria.