Forensic and clinical laboratories benefit from DNA standard reference materials (SRMs) that provide the quality control and assurance that their results from sequencing unknown samples are correct. Therefore, the mitochondrial DNA (mtDNA) genome of HL-60, a promyelocytic leukemia cell line, has been completely sequenced by four laboratories and will be available to the forensic and medical communities in the spring of 2003; it will be called National Institute of Standards and Technology (NIST) SRM 2392-I. NIST human mtDNA SRM 2392 will continue to be available and includes the DNA from two apparently healthy individuals. Both SRM 2392 and 2392-I contain all the information (e.g. the sequences of 58 unique primer sets) needed to use these SRMs as positive controls for the amplification and sequencing any DNA. Compared to the templates in SRM 2392, the HL-60 mtDNA in SRM 2392-I has two tRNA differences and more polymorphisms resulting in amino acid changes. Four of these HL-60 mtDNA polymorphisms have been associated with Leber Hereditary Optic Neuropathy (LHON), one as an intermediate mutation and three as secondary mutations. The mtDNA from a cell line (GM10742A) from an individual with LHON was also completely sequenced for comparison and contained some of the same LHON mutations. The combination of these particular LHON associated mutations is also found in phylogenetic haplogroup J and its subset, J2, and may only be indicative that HL-60 belongs to haplogroup J, one of nine haplogroups that characterize Caucasian individuals of European descent or may mean that haplogroup J is more prone to LHON. Both these mtDNA SRMs will provide enhanced quality control in forensic identification, medical diagnosis, and single nucleotide polymorphism detection.