Primers for vertebrate mitochondrial leading-strand DNA replication are products of transcription synthesized by mitochondrial RNA polymerase. The precursor primer RNA exists as a persistent RNA-DNA hybrid, known as an R-loop, formed during transcription through the replication origin (Xu, B., and Clayton, D. A. (1996) EMBO J. 15, 3135-3143). In an effort to examine the precise structure of this primer RNA intermediate, we have used two methods to reconstitute model R-loops containing the mouse mitochondrial DNA origin sequence. First, we demonstrate that bacteriophage SP6 RNA polymerase can efficiently catalyze the formation of an R-loop at the mouse mtDNA origin sequence. Second, the R-loop can be assembled by annealing presynthesized RNA and supercoiled DNA template in the presence of formamide. R-loop formation by either method is dependent on specific template sequences. The reconstituted R-loop is exceptionally stable and exhibits an unexpected structure. Structural studies indicate that the RNA strand is organized within the RNA-DNA base-paired region, suggesting that the heteroduplex interaction occurs through a specific conformation. We propose that the organized structure of the R-loop is critical for primer RNA function in vivo with important implications for the RNA processing and DNA replication machinery.