We have isolated and characterized the F0-ATPase subunit 6 gene (atp6) from tobacco mitochondria. The tobacco sequence exists as a single copy, is transcribed and contains an open reading frame (ORF) capable of encoding a peptide of 395 amino acids. The first 130 amino acids of the tobacco putative polypeptide show limited homology with the N terminus predicted for the maize ATPase subunit 6. Although poorly conserved at the sequence level, the tobacco and maize amino termini are hydrophilic and have a high percentage of charged amino acids. This portion of the predicted peptide may represent a presequence that is common to the ATPase subunit 6 of plants. Significant homology between tobacco and maize begins with amino acid 131, in a region that is highly conserved among fungal ATPase 6 subunits. In the remainder of the predicted protein, tobacco and maize share approximately 81% homology. A 41 bp sequence and a 175 bp conserved region found upstream from the tobacco atp6 coding region are homologous with sequence elements found in the 5' flanking regions of other plant mitochondrial genes and may be important for regulation and expression of the atp6 gene.