Extrachromosomal molecules of covalently closed cirular DNA (cccDNAs) were isolated from human fibroblasts near the end of their in vitro replicative lifespan and cloned into plasmid pBR322. Uncloned cccDNAs varied from several hundred to several thousand base pairs in size and contained a higher proportion of sequences homologous to the interspersed repetitive sequences AluI (SINES) and Kpnl (LINES), than to human alphoid and satellite III sequences that are tandemly repeated in the genome. After molecular cloning into pBR322, cccDNA inserts also showed a 3 to 4 fold over-representation of sequences homologous to Kpnl. There was also a strong age-dependent decline in the number of fibroblast RNA transcripts homologous to one of the cccDNAs containing a Kpnl sequence. The average size of cloned fibroblast cccDNAs was 2.52 kilobase pairs (Kbp) which is several fold larger than that reported for permanent mammalian cell lines. This may reflect fundamental differences in the mechanisms of generation of cccDNAs between mortal and immortal cells.