Animal mitochondrial DNA (mtDNA) maintains a displacement loop (D loop) at the heavy strand origin of replication. These D loops represent sharply limited synthesis of heavy strands and provide a unique opportunity to examine the termination of DNA synthesis. Direct sizing at the nucleotide level indicates that the 3' ends of D-loop strands of human and mouse mtDNA are discrete and map within three to five nucleotides on the complementary template strand. In the case of human mtDNA, there is a single trinucleotide stop point 51-53 nucleotides downstream from a 15-nucleotide template sequence (3'T-A-A-C-C-C-A-A-A-A-A-T-A-C-A 5') which is repeated four times in the mouse mtDNA D-loop region 3'(T-A-A-Py-Py-A-A-A-T-T-A-C-A 5'). The stop points of the five major mouse D-loop strands are 24-63 nucleotides downstream from the four repeated template sequences. These results suggest that the arrest of D-loop strand elongation is an event determined by template sequence.