Defects of the NADH dehydrogenase complex are predominantly manifested in mitochondrial diseases and are significantly associated with the development of many late onset neurological disorders such as Parkinson's disease. Here we describe an immunocapture procedure for isolating this multisubunit membrane-bound complex from human tissue. Using small amounts of immunoisolated protein, one-dimensional and two-dimensional gel electrophoresis, matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) peptide mass finger printing (PMF), and nanoflow liquid chromatography mass spectrometry/mass spectrometry (LC-MS/MS), we can resolve and identify the human homologues of 42 polypeptides detected so far in the more extensively studied beef heart complex I. These polypeptides include the GRIM-19 protein, which is claimed to be involved in apoptosis, a polypeptide first identified by gene screening as a neuronal protein, as well as a protein thought to be in differentiation linked processes. The concordance of data from human and bovine complex I isolated by different procedures adds to the certainty that these novel proteins of seemingly diverse function are a part of complex I.