Analysis of mitochondrial replication intermediates of Gallus gallus on fork-direction gels indicates that replication occurs in both directions around circular mitochondrial DNA. This finding was corroborated by a study of chick mitochondrial DNA on standard neutral two-dimensional agarose gels, which yielded archetypal initiation arcs in fragments covering the entire genome. There was, however, considerable variation in initiation arc intensity. The majority of initiation events map to regions flanking the major non-coding region, in particular the NADH dehydrogenase subunit 6 (ND6) gene. Initiation point mapping of the ND6 gene identified prominent free 5' ends of DNA, which are candidate start sites for DNA synthesis. Therefore we propose that the initiation zone of G. gallus mitochondrial DNA encompasses most, if not all, of the genome, with preferred initiation sites in regions flanking the major non-coding region. Comparison with mammals suggests a common mechanism of initiation of mitochondrial DNA replication in higher vertebrates.