Restriction endonuclease analysis of HeLa cells and cells in which origins have been questioned provides evidence in favor of a HeLa cell origin for the questioned cells. Digestion of cellular human DNA reveals a variable ribosomal DNA (rDNA) fragment that is present in up to four discrete sizes. Cell lines of known and suspected HeLa origin contain only two size variants. This pattern of variability serves to distinguish HeLa-derived cells from others. Despite repeated passage and divergence of the HeLa phenotype and karyotype, the restriction pattern is remarkably constant.