Human SCO1 and SCO2 are paralogous genes that code for metallochaperone proteins with essential, but poorly understood, roles in copper delivery to cytochrome c oxidase (COX). Mutations in these genes produce tissue-specific COX deficiencies associated with distinct clinical phenotypes, although both are ubiquitously expressed. To investigate the molecular function of the SCO proteins, we characterized the mitochondrial copper delivery pathway in SCO1 and SCO2 patient backgrounds. Immunoblot analysis of patient cell lines showed reduced levels of the mutant proteins, resulting in a defect in COX assembly, and the appearance of a common assembly intermediate. Overexpression of the metallochaperone COX17 rescued the COX deficiency in SCO2 patient cells but not in SCO1 patient cells. Overexpression of either wild-type SCO protein in the reciprocal patient background resulted in a dominant-negative phenotype, suggesting a physical interaction between SCO1 and SCO2. Chimeric proteins, constructed from the C-terminal copper-binding and N-terminal matrix domains of the two SCO proteins failed to complement the COX deficiency in either patient background, but mapped the dominant-negative phenotype in the SCO2 background to the N-terminal domain of SCO1, the most divergent part of the two SCO proteins. Our results demonstrate that the human SCO proteins have non-overlapping, cooperative functions in mitochondrial copper delivery. Size exclusion chromatography suggests that both the proteins function as homodimers. We propose a model in which COX17 delivers copper to SCO2, which in turn transfers it directly to the CuA site at an early stage of COX assembly in a reaction that is facilitated by SCO1.