We have constructed a cDNA library prepared from an hepatoma cell line (HTC cells) and isolated a clone, pHT 13, which corresponds to mRNAs present at a much higher level in rat hepatomas than in normal hepatocytes. The sequence of the pHT 13 insert has been previously published (Nucleic Acids Res. 1988, 16,10935). This clone contains mitochondrial DNA sequences with an abnormal organization, since it includes part of the NADH dehydrogenase subunit 6 (ND6) and of the cytochrome oxidase subunit I (COI) genes separated by 230 bases instead of 9 kb in mitochondrial genome from normal hepatocytes. In this work we show (1) that RNAs homologous to this clone are present in hepatoma cells but not in normal hepatocytes, (2) that a 3 kb fragment of tumor mitochondrial DNA contains both the ND6 and the COI sequences. The abnormal structure of the DNA is confirmed by Southern blot analysis which shows that distinct types of mitochondrial DNAs are present in hepatoma cells.