The organization of mitochondrial DNA was studied in seed-derived plants of sugar beet, Beta vulgaris L., and compared with the structure of DNA isolated from calli propagated in vitro. Tissues representing three genotypes (diploid male sterile, diploid maintainer and diploid fertile) were maintained under in vitro conditions for different periods before the DNA was analysed. Restriction fragment length polymorphism with BamHI and XhoI enzymes, and Southern hybridization with atpA and atp6 homologous probes were used. Hybridization experiments showed conspicuous differences in the organization of both genes in long-term callus cultures (2 years or older). The novel organization of mitochondrial DNA in the calli of the male sterile genotype showed an additional 3.9 kb fragment after hybridization with the atp6, and 7.9 kb and 3.5 kb bands after hybridization with atpA. In the fertile genotype, changes in mitochondrial DNA structure were manifested in the disappearance of a 2.1 kb fragment after probing with atpA. Alterations induced in the mitochondrial genome of the male sterile line showed unique features in mtDNA rearrangements.