The availability of the complete sequence of human mitochondrial DNA (mtDNA) has proven extremely useful in phylogenetic studies, forensic science and the determination of chimerism after allogeneic stem cell transplantation. In this study, we could demonstrate that the analysis of mtDNA polymorphisms is a quick and reliable method to identify contamination of human hematopoietic cell lines. This assay is based on PCR-sequencing of three hypervariable segments of the control region of mtDNA (hypervariable region (HRV) 1, 2 and 3). All three regions contain a large number of single-base polymorphisms. mtDNA was isolated according to standard laboratory procedures and amplified by PCR. Subsequently products were sequenced and evaluated with a semiautomated DNA sequencer system. So far, 21 human leukemia-lymphoma (LL) cell lines and nine other human cell lines were screened for contamination by other cell lines applying this method. We conclude that analysis of mtDNA polymorphisms is a quick, reliable and inexpensive method to detect intra - and interspecies cross-contamination and for the authentication of human LL cell lines. In comparison to other methods (cytogenetics, fluorescence in situ hybridization or immunophenotyping), this technique is less laborious and time consuming.