We have recently discovered a family of chromosomal mtDNA sequences, in several, but not all, felid species, that have a high degree of sequence homology to a large portion (8 kilobases [kb]) of the extrachromosomal mitochondrial (mt) DNA. This nuclear mitochondrial (Numt) DNA has been studied using molecular techniques as an approach to determining the molecular aspects of gene transposition, amplification and mtDNA evolution. Fractionation of cytoplasmic and nuclear DNAs, and further purification of supercoiled mtDNAs, revealed that these mtDNA sequences are located in a nuclear fraction. Chromosomal mapping using hybrid cell panels and fluorescent in situ hybridization indicate that Numt is located near the centromere of feline chromosome D2. Restriction enzyme mapping analysis of the cloned Numt DNA sequence suggested the tandemly repeated structure of 7.8-kb Numt DNA is a single unit. Pedigree analysis by pulse field gel electrophoresis of high molecular weight DNAs digested with three, six-cutter restriction enzymes revealed Numt DNA bands ranging from 240 to over 600 kb which segregate in a Mendelian fashion. These results suggest that Numt DNA is clustered to a single locus as an mtDNA fragment tandemly amplified at least 40 times. Sequence analysis of the Numt 7.8-kb clone confirmed the existence of genes for 12S and 16S ribosomal RNAs, transfer RNAs of serine, aspartate, isoleucine, cytochrome oxidase I and II (COI, II), and NADH dehydrogenase 1 and 2 with the same gene order as mtDNA. However, the COII gene sequence was connected with D-loop regions and 12S rRNA sequences. Thus, Numt DNA contains approximately one half of the genome of intact mtDNA. On the border between COII and D-loop regions, direct five-time repeats of the motif, ACACACGT were found. This evidence may suggest the involvement of this repeat sequence in the occurrence of Numt in the domestic cat.