We demonstrate the use of immobilized metal ion affinity chromatography for isolating the constituent subunits of assembled mitochondrial ATP synthase (mtATPase) wherein a single subunit of the complex has been modified to contain hexahistidine. Genes encoding subunit d or OSCP of mtATPase from Saccharomyces cerevisiae were modified each to encode a polypeptide having a C-terminal addition of six consecutive histidines. Expression of plasmid- borne modified genes, in host yeast cells lacking a functional copy of the relevant endogenous gene, generated functional mtATPase complexes as judged by growth of rescued cells on the nonfermentable substrate ethanol. Significantly, the oligomycin-sensitive ATP hydrolase activity in mitochondria from cells expressing tagged subunits was similar to that of cells expressing unmodified subunits, indicating that there had been no impairment of the functional integrity of mtATPase. Mitochondrial lysates were prepared from each strain and subjected to chromatography under nondenaturing conditions on a resin containing immobilized Ni2+. It is likely that the mtATPase complexes adsorbed by immobilized metal ion affinity chromatography are fully assembled because their subunit composition closely matches that of a preparation of assembled mtATPase conventionally isolated from mitochondrial lysates by ammonium sulfate precipitation and purification by sucrose gradient centrifugation. Furthermore, assembled mtATPase containing a tagged subunit could be adsorbed, albeit at lower yield, when the relevant modified gene was expressed in wild-type host cells. The general application of this novel isolation procedure greatly simplifies and reduces the number of steps required for the isolation of assembled multi-subunit complexes. Moreover, the approach may be used for studying subunit-subunit interactions within the mtATPase complex.