A fragment containing non-heme iron and acid-labile sulfide but little flavin can be solubilized from the mitochondrial NADH-ubiquinone oxidoreductase complex with chaotropic agents. This iron-protein fragment [Hatefi, Y., & Stempel, K. E. (1969) J. Biol. Chem. 244, 2350] has been resolved with detergents and ammonium sulfate fractionation into iron and acid-labile sulfide containing fractions, here called ISP-I and ISP-(II + III). ISP-I consists predominantly of a single polypeptide of molecular weight 75000. ISP-(II + III) consists predominantly of three polypeptides in equimolar concentrations with molecular weights of 49,000, 30000, and 13000. Treatment of the latter with sodium trichloroacetate followed by ammonium sulfate fraction results in separation of the 49000 molecular weight polypeptide from the two smaller subunits. Both of these subfractions (ISP-II and ISP-III, respectively) contain non-heme iron. The three iron-sulfur proteins have been characterized by their absorption spectra and iron and acid-labile sulfide contents. On the basis of the distribution of iron among the fractions obtained from chaotropic resolution of the NADH-ubiquinone oxidoreductase complex, a minimum of six or seven iron-sulfur centers are present in this enzyme.