Mammalian mitochondrial DNA often contains a short DNA displacement loop at the heavy-strand origin of replication. This short nascent DNA molecule has been used to study site-specific termination of mitochondrial DNA synthesis in human and mouse cells. We examined D-loop strand termination in two distantly related artiodactyls, the pig and the cow. Porcine mitochondrial DNA was unique among mammals in that it contained only a single species of D-loop single-stranded DNA. Its 3' end mapped to a site 187 nucleotides from the 5' end of the proline tRNA gene. This site was 21 and 47 nucleotides 5' to two very similar sequences (5' ACATATPyATTAT 3') which are closely related to the human and mouse termination-associated sequences noted by Doda et al. (J. N. Doda, D. T. Wright, and D. A. Clayton, Proc. Nat. Acad. Sci. USA 78:616-6120, 1981). Bovine mitochondrial DNA contained three major D-loop DNA species whose 3' ends mapped to three different sites. These sites were not found in the porcine sequence. However, the bovine termination sites were located 60 to 64 base pairs 5' from sequences which were also very similar to the termination-associated sequences present in pigs and other mammals. These results firmly establish the concept that arrest of heavy-strand DNA synthesis is an event determined, at least in part, by template sequence. They also suggest that arrest is determined by sequences which are a considerable physical distance away from the actual termination site.Mitochondrial DNA (mtDNA) in mammalian cells exists as a covalently closed, circular, supercoiled, doublestranded DNA molecule approximately 16,000 base pairs in length (13). Each strand is replicated unidirectionally from a separate origin. Although synthesis of daughter heavy (H) strands begins at discrete genome locations, the precise nucleotide sequence requirements for H-strand initiation have not yet been determined and appear to vary considerably among mammals (10). After initiation, most newly initiated H strands terminate <700 nucleotides downstream. This leads to a partial relaxation of parental supercoiled molecules because the short, newly synthesized H strand, variously termed D-loop strand (11), 12,15,19), or DH-DNA (7-9), remains associated with the template, thus creating a triple-stranded structure known as a displacement loop (D-loop) molecule. The major form of mammalian mtDNA isolated from cells is this triplestranded, partially replicated D-loop molecule (10).Previous studies mapping the 3' ends of D-loop strands suggested that a portion of the template sequence may be involved in the arrest of H-strand synthesis within the D-loop region. D-loop molecules isolated from mouse L cells contain four major D-loop strands with four distinct 3' ends (11), while those of human HeLa cells contain three major D-loop strands but only one major 3' end (12). The existence of a conserved nucleotide sequence approximately 30 to 60 nucleotides from each 3' end prompted Doda et al. (11) to propose that this sequence may play a role in determ...