To maintain and to differentiate into various plastid lineages, replication of the plastid DNA (ptDNA) and division of the plastid must take place. However, replication initiation of the ptDNA has been less understood. The present study describes identification of the initiation region (origin) of ptDNA replication in the rice cultured cells. RNA-primed newly replicated DNA strands pulse-labeled with bromodeoxyuridine were isolated and size-fractionated. Locations of these nascent strands on the ptDNA determined the two major origin regions around the 3′ region of each 23S rDNA in the inverted repeats (IRA and IRB). Two-dimensional agarose gel electrophoresis of the replication intermediates suggested that replication from each origin proceeds bidirectionally. This contrasted to replication by the double D - loop mechanism.