- Nuclear mitochondrial DNA sequences (NUMTs) are common in eukaryotes. However, the mechanism by which they integrate into the nuclear genome remains a riddle. We analyzed 247 NUMTs in the human nuclear DNA (nDNA), along with their flanking regions. This analysis revealed that some NUMTs have accumulated many changes, and thus have resided in the nucleus a long time, while others are >94% similar to the reference human mitochondrial DNA (mtDNA), and thus must be recent. Among the latter, two NUMTs, encompassing the COI gene, carry a set of transitions characteristic of the extant African-specific L macrohaplogroup mtDNAs and are more homologous to human mtDNA than to chimp. Screening for one of these NUMTs revealed its presence in all human samples tested, confirming that the African macrohaplogroup L mtDNAs were present in the earliest modern humans and thus were the first human mtDNAs. An analysis of flanking sequences of the NUMTs revealed that 59% were within 150 bp of repetitive elements, with 26% being within 15 bp of and 33% being within 15–150 bp of repetitive elements. Only 14% were integrated into a repetitive element. This association of NUMTs with repetitive elements is highly nonrandom (p<0.001). These data suggest that the vicinity of transposable elements influences the ongoing integration of mtDNA sequences and their subsequent duplication within the nDNA. Finally, NUMTs appear to preferentially integrate into DNA with different GC content than the surrounding chromosomal band. Our results suggest that chromosomal structure might influence integration of NUMTs. Hum Mutat 23:125–133, 2004. © 2003 Wiley-Liss, Inc.