This paper deals with the relationship of chloroplast DNA (ctDNA) and cytoplasmic malesterility (CMS) of maize, wheat and rape. Intramolecular heterogeneity and fragment patterns of DNA digested with restriction endonucleases were compared and analyzed. For this purpose methods of agarose gel electrophoresis and two dimensional gel electrophoresis with denaturating solvent concentration gradients were applied. 1) Heat denaturation of ctDNA from a male sterile line in maize yielded a profile with three melting regions. This base sequence heterogeneity was not observed for the ctDNA of its maintainer. 2) Maize ctDNA was digested with EcoR1 and BamH1 restriction endonucleases, wheat and rape ctDNA with EcoR1 only. No significant differences were observed in the comparison of the fragment patterns from the sterile lines and their maintainers with the exception of the sterile line in rape which lacked one fragment present in its maintainer. 3) Two dimensional electrophoresis using a gradient of denaturing agents revealed several differences in number and relative positions of the separated restriction endonuclease fragments between the sterile lines and their respective maintainers in all three tested crops. This raises the possibility that changes in ctDNA may be involved in CMS.