In solid tissues of vertebrates, initiation of mitochondrial DNA replication encompasses a broad zone downstream of the major noncoding region (NCR). In contrast, analysis with two-dimensional agarose gel electrophoresis of mitochondrial DNA replication intermediates in cultured human cells revealed initiation concentrated in the NCR. Mapping of prominent free 5' ends on the heavy strand of mitochondrial DNA identified two clusters of potential start sites. One mapped to the previously assigned origin of strand-asynchronous replication (O(H)); the other lay several hundred nucleotides away from O(H), toward the other end of the NCR. The latter cluster is proposed to be the major site of bidirectional replication initiation on the basis of the following: its prominence is enhanced in cells amplifying mitochondrial DNA after experimentally induced mitochondrial DNA depletion; free 5' ends are found in corresponding positions on the opposite strand; it is transient in nature; and it is associated with bubble arcs.