Three of the respiratory chain complexes contain essential iron-sulfur (Fe/S) cluster prosthetic groups. Besides respiration, these ancient inorganic cofactors are also necessary for numerous other fundamental biochemical processes in virtually every known organism. Both the synthesis of Fe/S clusters and their delivery to apoproteins depend on the concerted function of specialized, often dedicated, proteins located in the mitochondria and cytosol of eukaryotes. Impaired function of the mitochondria-located Fe/S cluster (ISC) assembly machinery affects all cellular Fe/S proteins, including enzymes of the respiratory chain, NADH: ubiquinone oxidoreductase (complex I; eight Fe/S clusters), succinate: ubiquinone oxidoreductase (complex II; three Fe/S clusters), and cytochrome bc(1) complex (complex III; one Fe/S cluster). Here, we describe strategies and techniques both to deprive respiratory chain proteins of their Fe/S cofactors and to study changes in activity and composition of these proteins. As examples, we present the results of the depletion of two types of Fe/S biogenesis proteins, huNfs1 and huInd1, in a human tissue culture model. The ISC assembly component huNfs1 is required for biogenesis of all cellular Fe/S proteins, its loss exerting pleiotropic effects, whereas huInd1 is specific for Fe/S cluster maturation of complex I. Disorders in Fe/S cluster assembly are candidate causes for defects in respiratory complex assembly of unknown etiology.