The petunia mitochondrial fused gene (pcf), which is associated with cytoplasmic male sterility (CMS), is composed of sequences derived from atp9, coxII, and an unidentified reading frame termed urfS. Pcf transcripts are modified by editing at 11 sites. Codon usage and nearest neighbor analysis suggest that the urfS region is not derived originally from a plant mitochondrial coding region. Although the gene contains an open reading frame coding for a 43 kDa protein, a 25 kDa gene product has previously been identified (Nivison and Hanson, 1989). N-terminal sequencing revealed that the 25 kDa protein is encoded within the urfS portion of pcf and that its actual molecular mass is 19.5 kDa. Through pulse-chase labeling of protein in isolated mitochondria, the 25 kDa protein was found to be processed from a 43 kDa precursor protein representing the entire pcf gene sequence. Antibodies to synthetic peptides encoded by the atp9 and coxII portions of pcf recognized petunia ATP9 or COXII but no other mitochondrial proteins on immunoblots. Controlled proteolysis experiments showed that both the 43 kDa precursor and the 25 kDa protein are soluble or loosely associated with membranes. Thus, the 25 kDa protein appears to be the only pcf-encoded protein that accumulates in mitochondria.