Brain mtDNA from rats ranging in age from 1 day to 33 months were analyzed for large-scale rearrangements using nested PCR. The region of the mtDNA targeted by the primers was the shorter arc between the two orgins of replication and encompassed the heavy (H) and light (L) strand promoters (HSP) and (LSP). Rearrangements lacking 4 to 5 kb of genomic sequence were found in animals of all ages. Twenty-two different rearrangements were sequenced; two of these were found replicated in several different animals. All the rearrangements identified lacked an HSP and six lacked an LSP as well. The end points of each rearrangement had short direct repeats of 9 bp or less, but repeats of 4 bp or less were the most common. The mode of involvement of the direct repeats in the rearrangement mechanism varied since in some cases a sequence precisely equivalent to one member of the paired repeats was found at the junction; whereas in other cases, more or less than one complete member was found. Sixteen of the 22 rearrangements terminated on one side within a 22-bp locus, or hot spot, located at a potential stem-loop structure midway between the HSP and LSP. The other ends of these rearrangements were at different sites. In addition, a secondary hot spot was found near the junction between the tRNA Ala and tRNA Asn genes, which lie in a cluster of five tRNA genes that surround the stem-loop structure of the L-strand origin of replication. The data suggest a link between secondary structure and short direct repeats and the rearrangement mechanism(s). The results of this study, in conjunction with our previous study of the longer arc of rat mtDNA (Van Tuyle, G.C., J.P. Gudikote, V.H. Hurt, B.B. Miller and C.A. Moore (1996) Multiple, Large deletions in rat mitochondrial DNA: Evidence for a major hot spot, Mutation Res., 349, 95–107), indicate that nearly the entire mitochondrial genome is subject to rearrangement mutations that are detectable in brain tissue throughout an animal's life span.