The mitochondrial genome of the Polima (pol) male-sterile cytoplasm of Brassica napus contains a chimeric 224-codon open reading frame (orf224) that is located upstream of, and co-transcribed with, the atp6 gene. The N-terminal coding region of orf224 is derived from a conventional mitochondrial gene, orfB, while the origin of the remainder of the sequence is unknown. We show that an apparently functional copy of orfB is present in the pol mitochondrial genome, indicating that the pol CMS is not caused by the absence of an intact, expressed orfB gene. The 5' termini of orf224/atp6 transcripts present in both sterile and fertility-restored (Rf) pol cytoplasm plants are shown to map to sequences resembling mitochondrial transcription-initiation sites, whereas the 5' termini of two transcripts specific to restored lines map to sequences which resemble neither one another nor mitochondrial promoter motifs. It is suggested that the complex orf224/atp6 transcript pattern of Rf plants is generated by a combination of multiple transcription initiation and processing events and that the nuclear restorer gene acts to specifically alter orf224/atp6 transcripts by affecting RNA processing. Northern analyses demonstrate that the effect of the restorer gene on orf224/atp6 transcripts is not tissue or developmental-stage specific. However, the expression of the atp6 region is developmentally regulated in pol plants, resulting in decreased levels of monocistronic atp6 transcripts in floral tissue relative to seedlings. It is suggested that this developmental regulation may be related to the absence of overt phenotypic effects of the CMS mutation in vegetative tissues.