The respiratory complex I of mitochondria consists of some 40 different subunits which form an L-shaped structure. Perpendicular to a hydrophobic arm embedded in the inner mitochondrial membrane a peripheral arm protrudes into the matrix. Assembly of the complex as studied in the fungus Neurospora crassa involves the formation of discrete intermediates. The matrix arm and the membrane arm are formed independently of each other and are joined in the course of assembly. The membrane arm itself is formed by association of two assembly intermediates, a smaller of 200 kDa and a larger of 350 kDa. The latter is associated with two extra proteins of 84 and 30 kDa which are not constituent parts of mature complex I. Their primary structures show no similarity to known proteins. Mutants generated by disrupting the genes of either of the two proteins accumulate the matrix arm of complex I and the small membrane arm assembly intermediate, but are incapable of forming the large intermediate. In the wild-type, the extra proteins exclusively associate with the large membrane arm assembly intermediate. Pulse-chase labelling experiments showed that the two proteins are repeatedly involved in many assembly cycles of the intermediate. These results indicate that the two proteins are novel chaperones specific for complex I membrane arm assembly.