The previously detected Rieske iron-sulfur protein from the membranes of the thermoacidophile Sulfolobus acidocaldarius [Anemüller, S., et al. (1993) FEBS Lett. 318, 61-64] was purified to electrophoretic homogeneity and the N-terminal amino acids determined. The apparent molecular weight was estimated to be 32 kDa. The reduced protein displays a rhombic EPR spectrum with gxyz = 1.768, 1.895, 2.035. The average g-value of 1.902 is typical for nitrogen ligand-containing clusters. EPR spin quantification and the iron content indicate the presence of one [2Fe-2S] cluster. The purified protein displays ubiquinol cytochrome c reductase activity. The pH optimum of this reaction is temperature dependent and was determined to be pH 7 at 56 degrees C. The results presented in this study clearly prove that the Sulfolobus Rieske protein belongs to the family of the true Rieske iron-sulfur proteins.