To identify regions of the mitochondrial genome from the polima or pol male-sterile cytoplasm of Brassica napus that are genetically correlated with cytoplasmic male sterility (CMS) we analyzed mtDNAs of three male-sterile somatic hybrids formed by the fusion of broccoli (B. oleracea L. var. italica) and pol CMS B. napus protoplasts. Fragments characteristic of a 4.5 kb DNA segment that comprises the single organizational difference between sterile pol and fertile cam Brassica mitochondrial genomes were found in all three sterile somatic hybrids. One of these hybrids possessed a mitochondrial genome that was, apart from a limited region around this 4.5 kb CMS-associated segment, collinear with B. oleracea mtDNA. Previous studies have indicated that expression of transcripts spanning the atp6 gene and a chimeric gene, orf24, located on this 4.5 kb DNA segment, is associated with male sterility. The present results indicate that the orf224/atp6 gene region is genetically correlated with male sterility and provide significant additional support for the view that this gene region may be involved in specifying the CMS trait.