Characteristic rearranged human chromosome markers have been observed in a variety of HeLa cell sublines and in five suspected HeLa contaminant lines originally thought to be derived from differentiated tissues of different individual patients. The allozyme genetic signatures, representing the composite enzyme phenotype at eight polymorphic loci, of each of the studied contaminant lines were identical to each other and to those of HeLa cells. The probability that each of these lines would have an identical genetic signature (since the frequency of the HeLa genotype is 0.0017) is 4.2 X 10(-15). Differences between cell lines, however, could be detected by isoelectric focussing of the isoenzymes for glucose-6-phosphate dehydrogenase. The cell lines, including CLL 74, the older Chang liver line, failed to express five liver-specific proteins. One protein was detected in a new liver cell culture. Variations in cytogenetic, biochemical, and differentiated functions during continuous cell culture are discussed.