Divalent metal binding proteins in the Arabidopsis mitochondrial proteome were analysed by mobility shifts in the presence of divalent cations during two-dimensional diagonal sodium dodecyl sulphate-polyacrylamide gel electrophoresis. Tandem mass spectrometry and searches of the predicted Arabidopsis protein dataset were used in an attempt to identify 34 of the proteins which shifted. This analysis identified a total of 23 distinct protein spots as the products of at least 11 different Arabidopsis genes. A series of proteins known to be divalent cation-binding proteins, or to catalyse divalent cation-dependent reactions, were identified. These included: succinyl CoA ligase beta subunit, Mn-superoxide dismutase (SOD), an Fe-S centred component of complex I and the REISKE iron-sulphur protein of the b/c(1) complex. A further set of four proteins of known function but without known divalent binding properties were also identified: the Vb subunit of cytochrome c oxidase, a subunit of ATP synthase (orfB), the acyl carrier protein, and the translocase of the outer membrane (TOM20). Three other proteins, of unknown function, were also found to shift in the presence of divalent cations. This approach has broad application for the identification of sub-proteomes based on the metal interaction of polypeptides.