With 46 subunits, human mitochondrial complex I is the largest enzyme of the oxidative phosphorylation system. We have studied the assembly of complex I in cultured human cells. This will provide essential information about the nature of complex I deficiencies and will enhance our understanding of mitochondrial disease mechanisms. We have found that 143B206 rho zero cells, not containing mitochondrial DNA, are still able to form complex I subcomplexes. To further address the nature of these subcomplexes, we depleted 143B osteosarcoma cells of complex I by inhibiting mitochondrial protein translation with doxycycline. After removing this drug, complex I formation resumes and assembly intermediates were observed by two-dimensional blue native electrophoresis. Analysis of the observed subcomplexes indicates that assembly of human complex I is a semi-sequential process in which different preassembled subcomplexes are joined to form a fully assembled complex. The membrane part of the complex is formed in distinct steps. The B17 subunit is part of a subcomplex to which ND1, ND6 and PSST are subsequently added. This is bound to a hydrophilic subcomplex containing the 30 and 49 kDa subunits, to which a subcomplex including the 39 kDa subunit is incorporated, and later on the 18 and 24 kDa subunits. At a later stage more subunits, including the 15 kDa, are added and holo-complex I is formed. Our results suggest that human complex I assembly resembles that of Neurospora crassa, in which a membrane arm is formed and assembled to a preformed peripheral arm, and support ideas about modular evolution.