Soybean suspension culture cells were subjected to mutagenesis with ethyl methane sulfonate and cells resistant to the ATP synthase inhibitor oligomycin were recovered. Suspension cultures showed high levels of resistance over a period of 17 months after mutagenesis. The level of resistance to oligomycin decreased during prolonged growth in the absence of the selection agent. Although mitochondrial inheritance of the resistance phenotype was not tested, this observation is consistent with segregation of resistant and sensitive mitochondria in progeny cells. Cross resistance to venturicidin was also observed which suggested the possible involvement of the mitochondrial ATP synthase subunit 9 gene, however, sequences of ATP synthase subunit 9 cDNA clones from resistant cells were identical to the wild type sequence. In addition, Southern blot analyses of DNA from wild type and resistant cultures did not identify rearrangements. These results indicated that resistance to oligomycin could not be directly attributed to a mutation in the primary gene sequence, an alteration in the RNA editing pattern of transcripts, or gross rearrangements in the region containing and directly adjacent to the ATP synthase subunit 9 gene in the mitochondrial DNA from resistant cultures.