HindIII digestion of chloroplast DNA (ctDNA) from normal (N) and male sterile (S) lines of sugar beet generated 26 (N ctDNA) and 25 (S ctDNA) discrete bands ranging in size from 0.45×106 to 14.5×106 daltons (d). Densitometric tracings and a Southern hybridization experiment revealed one band (H7) missing and replacement of this band by two fragments (H12b and H14b) in S ctDNA. A slight intraspecific heterogeneity is likely to be due to the creation of a new HindIII site in the left part of the large single-copy region of S ctDNA. The summation of HindIII fragments indicated a molecular weight of 97.38 (N ctDNA) or 97.53 (S ctDNA) ×106 d. Southern hybridization analyses demonstrated that the chloroplast ribosomal RNA genes are located on three HindIII fragments of H4, H20 and H22, the large subunit (LS) gene of ribulose-1, 5-bisphosphate carboxylase/oxygenase on H6 and H17 fragments, the ribosomal protein CS-19 gene on 112 and H5a fragments and the 32, 000 d thylakoid protein gene on a H5b fragment. Twelve (derived from N ctDNA) and 18 (derived from S ctDNA) HindIII fragments have been cloned into pBR 322.