The F(1) component of mitochondrial ATP synthase is an oligomeric assembly of five different subunits, alpha, beta, gamma, delta, and epsilon. In terms of mass, the bulk of the structure ( approximately 90%) is provided by the alpha and beta subunits, which form an (alphabeta)(3) hexamer with adenine nucleotide binding sites at the alpha/beta interfaces. We report here ultrastructural and immunocytochemical analyses of yeast mutants that are unable to form the alpha(3)beta(3) oligomer, either because the alpha or the beta subunit is missing or because the cells are deficient for proteins that mediate F assembly (e.g. Atp11p, Atp12p, or Fmc1p). The F(1) alpha(1) and beta subunits of such mutant strains are detected within large electron-dense particles in the mitochondrial matrix. The composition of the aggregated species is principally full-length F(1) alpha and/or beta subunit protein that has been processed to remove the amino-terminal targeting peptide. To our knowledge this is the first demonstration of mitochondrial inclusion bodies that are formed largely of one particular protein species. We also show that yeast mutants lacking the alpha(3)beta(3) oligomer are devoid of mitochondrial cristae and are severely deficient for respiratory complexes III and IV. These observations are in accord with other studies in the literature that have pointed to a central role for the ATP synthase in biogenesis of the mitochondrial inner membrane.