Bovine-heart NADH:ubiquinone oxidoreductase (EC 1.6.5.3; Complex I) is the first and most complicated enzyme in the mitochondrial respiratory chain. Biochemistry textbooks and virtually all literature on this enzyme state that it contains one FMN and at least four iron-sulfur clusters. We show here that this statement is incorrect as it is based on erroneous protein determinations. Quantitative amino acid analysis of the bovine Complex I, to our knowledge the first reported thus far, shows that the routine protein-determination methods used for the bovine Complex I overestimate its protein content by up to twofold. The FMN content of the preparations was determined to be at least 1.3-1.4 mol FMN/mol Complex I. The spin concentration of the electron paramagnetic resonance (EPR) signal ascribed to iron-sulfur cluster N2 was determined and accounted for 1.3-1.6 clusters per molecule of Complex I. These results experimentally confirm the hypothesis [FEBS Lett. 485 (2000) 1] that the bovine Complex I contains two FMN groups and two clusters N2. Also the protein content of preparations of the soluble NAD(+)-reducing [NiFe]-hydrogenase (EC 1.12.1.2) from Ralstonia eutropha, which shows clear evolutionary relationships with Complex I, scores too high by the colorimetric protein-determination methods. Determination of the FMN content and the spin concentration of the EPR signal of the [2Fe-2S] cluster shows that this hydrogenase also contains two FMN groups. A third enzyme (Ech), the membrane-bound [NiFe]-hydrogenase from Methanosarcina barkeri which shows an even stronger evolutionary relationship with Complex I, behaves rather normal in protein determinations and contains no detectable acid-extractable FMN in purified preparations.