Surf1p is a protein of the inner membrane of mitochondria that functions in the assembly of cytochrome-c oxidase. The specifics of the role of Surf1p have remained unresolved. Numerous mutations in human Surf1p lead to severe mitochondrial disease. A homolog of human Surf1p is encoded by the genome of the ␣-proteobacterium Rhodobacter sphaeroides, which synthesizes a mitochondrial-like aa 3 -type cytochrome-c oxidase. The gene for Surf1p was deleted from the genome of R. sphaeroides. The resulting aa 3 -type oxidase was purified and analyzed by biochemical methods plus optical and EPR spectroscopy. The oxidase that assembled in the absence of Surf1p was composed of three subpopulations with structurally distinct heme a 3 -Cu B active sites. 50% of the oxidase lacked heme a 3 , 10 -15% contained heme a 3 but lacked Cu B , and 35-40% had a normal heme a 3 -Cu B active site with normal activity. Cu A assembly was unaffected. All of the oxidase contained low-spin heme a, but the environment of the heme a center was slightly altered in the 50% of the enzyme that lacked heme a 3 . Introduction of a normal copy of the gene for Surf1p on an exogenous plasmid resulted in a single population of normally assembled, highly active enzyme. The data indicate that Surf1p plays a role in facilitating the insertion of heme a 3 into the active site of cytochrome-c oxidase. The results suggest that maturation of the heme a 3 -Cu B center is a step that limits the association of subunits I and II in the assembly of mitochondrial cytochrome oxidase.