mit- mutants with genetically defined mutations in the mitochondrial structural genes of the H+-ATPase membrane subunits 6, 8 and 9 were analysed to determine the H+-ATPase assembly defects that resulted as a consequence of the mutations. These include mutants which do not synthesize one of the membrane subunits and mutants which can synthesize these subunits, but in an altered form. Protein subunits which can still be assembled to the defective H+-ATPase in these mutants were determined by immunoprecipitation using a monoclonal antibody to the beta-subunit of the enzyme complex. The results suggest that the assembly pathway of the mitochondrially synthesized H+-ATPase subunits involves the sequential addition of subunits 9, 8 and 6 to a membrane-bound F1-sector. In addition to subunits of the F0- and F1-sectors, two other polypeptides (Mr = 18,000 and Mr = 25,000) are associated with the yeast H+-ATPase. These polypeptides were not observed in the immunoprecipitates obtained from mutants in which the F0-sector is not properly assembled.