In Nicotiana sylvestris, two cytoplasmic male sterile (CMS) mutants obtained by protoplast culture show abnormal developmental features of both vegetative and reproductive organs, and mitochondrial gene reorganization following homologous recombination between 65 bp repeated sequences. A mitochondrial region of 16.2 kb deleted from both CMS mutants was found to contain the last two exons of the nad7 gene coding for a subunit of the mitochondrial respiratory chain complex I, which is encoded in the nucleus in fungi and animals but was recently found to be encoded by the mitochondrial genome in wheat. Although the N. sylvestris nad7 gene shows strong homology with its wheat counterpart, it contains only three introns instead of four. Polymerase chain reaction (PCR) experiments indicated that the parental gene organization, including the complete nad7 gene, is probably maintained at a substoichiometric level in the CMS mutants, but this proportion is too low to have a significant physiological role, as confirmed by expression studies showing the lack of detectable amounts of the NAD7 polypeptide. Consequently, absence of NAD7 is not lethal to plant cells but a deficiency of complex I could be involved in the abnormal CMS phenotype.