Alloplasmic male sterility is commonly obtained in Nicotiana by combining the nucleus from N. tabacum with the cytoplasm from other Nicotiana species. Besides being male-sterile, most of these cultivars also display changes in floral organ structure. Flowers from male-sterile plants containing the nucleus from N. tabacum combined with N. repanda cytoplasm develop stamen with shortened filaments and shrivelled anthers capped with stigmatoids. Male fertility and normal floral development can be restored by introduction of a restorer chromosome fragment from the cytoplasmic donor N. repanda into the N. tabacum nucleus. A novel reading frame, orf274, located upstream of atp1 in the mitochondrial genomes of both N. tabacum and N. repanda, as well as in the male-sterile and fertility-restored plants was identified. Co-transcripts of orf274 and atp1 were detected by RT-PCR in all four cultivars, but these transcripts accumulate to levels detectable by northern hybridization only in male-sterile plants. These co-transcripts neither generate detectable levels of an ORF274 polypeptide nor lead to altered expression of the ATP synthase subunit alpha. Measurement of ATP and ADP steady-state levels, however, revealed that the ATP/ADP ratio is significantly lower in young floral buds of male-sterile plants than in fertile plants.