The contamination of HeLa cells into many culture cells has been suspected. We detected HPV type 18 DNA in two HeLa cell sublines, Hep-2 laryngeal cancer and KB oral cancer cell lines as well as FL cell line derived from normal amniotic membrane which have been extensively used so far in experimental virology. On the other hand, we detected HPV type 18 DNA neither in other epithelial cell lines nor in fibroblastic human embryonic cells. The digestion analysis with several restriction enzymes of the HPV type 18 DNA in HeLa, Hep-2, KB and FL cell lines were almost the same. The molecular size of episomal DNA could not be demonstrated by digestion of these DNAs with XhoI, a non-cut enzyme for HPV type 18 DNA, suggesting all HPV types 18 DNA were integrated. Interestingly enough, the HPV type 18 specific DNA in these cells was cleaved into two fragments with another non-cut enzymes, B g/II and HindIII, suggesting the presence of a single cutting site for each enzymes presumably generated by rearrangements or mutations. These results provide further evidence that the so-called Hep-2, KB and FL cells are all HeLa cell derivatives.