Complex I (NADH:ubiquinone oxidoreductase) purified from bovine heart mitochondria was treated with the detergent N,N-dimethyldodecylamine N-oxide (LDAO). The enzyme dissociated into two known subcomplexes, Iα and Iβ, containing mostly hydrophilic and hydrophobic subunits, and a previously undetected fragment referred to as Iγ. Subcomplex Iγ contains the hydrophobic subunits ND1, ND2, ND3, and ND4L which are encoded in the mitochondrial genome, and the nuclear-encoded subunit KFYI. During size-exclusion chromatography in the presence of LDAO, subcomplex Iα lost several subunits and formed another characterized subcomplex known as Iλ. Similarly, subcomplex Iβ dissociated into two smaller subcomplexes, one of which contains the hydrophobic subunits ND4 and ND5; subcomplex Iγ released a fragment containing ND1 and ND2. These results suggest that in the intact complex subunits ND1 and ND2 are likely to be in a different region of the membrane domain than subunits ND4 and ND5. The compositions of the various subcomplexes and fragments of complex I provide an organization of the subunits of the enzyme in the framework of the known low resolution structure of the enzyme.