Covalently closed circular DNA molecules (cccDNA) from the human HeLa cell line were purified (96% pure by weight) by use of ATP-dependent deoxyribonuclease, and cloned into the HindIII site of phage lambda vector Charon 7. From the cccDNA library thus obtained, nine recombinants carrying mitochondrial DNA and 36 recombinants carrying small polydisperse circular (spc) DNA were picked at random for subsequent tests. The inserted fragments of spcDNA ranged in size from 0.6 to 7.6 kb with a mean length of 1.9 kb, a value which is the same as the average length of spcDNA. Analysis of the cloned spcDNA fragments revealed that (a) all the spcDNA clones investigated shared homologies with chromosomal DNA sequences, (b) all but one cloned DNA contained repetitive sequences, (c) the sequence organization could be roughly classified according to the reiteration frequency as greater than 10(5) (Alu family class), 10(4) to 10(5) (KpnI family class), 10(3) to 10(4) (mitochondrial DNA class) and less than 10(3) times per haploid genome, and (d) most of the repetitive sequences were dispersed in the genome, although some appeared clustered.