The redox properties of the [2Fe-2S] cluster in the 24 kDa subunit of bovine heart mitochondrial NADH:ubiquinone oxidoreductase (complex I) and three of its homologues have been defined using protein-film voltammetry. The clusters in all four examples display characteristic, pH-dependent redox transitions, which, unusually, can be masked by high ionic strength conditions. At low ionic strength (10 mM NaCl) the reduction potential varies by approximately 100 mV between high and low pH limits (pH 5 and 9); thus the redox process is not strongly coupled and is unlikely to form part of the mechanism of energy transduction in complex I. The pH dependence was shown to result from pH-linked changes in protein charge, due to nonspecific protonation events, rather than from the coupling of a specific ionizable residue, and the ionic strength dependence at high and low pH was modeled using extended Debye-Hückel theory. The low potential of the 24 kDa subunit [2Fe-2S] cluster, out of line with the potentials of the other iron-sulfur clusters in complex I, is suggested to play a role in coupling reducing equivalents at the catalytic active site. Finally, the validity of using the [2Fe-2S] cluster in an isolated subunit, as a mechanistic basis for coupled proton-electron transfer in intact complex I, is evaluated.