A partially purified replicative system of pea chloroplast that replicates recombinant DNAs containing pea chloroplast origin sequences has been characterised. Polymerisation by this system is very fast and insensitive to chain terminators like dideoxynucleotides, arabinosylcytosine 5'-triphosphate, etc. Both strands of template DNA are synthesized and single-stranded DNA templates undergo more than one round of replication.When sequences of either of the two chloroplast origins of replication (OriA or OriB) are used as templates, the replicative intermediates are found to have sigma structures. Electron microscopic analysis of the sigma structures restricted with various enzymes reveals that the initiation site of in vitro replication maps near the displacement-loop regions where replication initiates also in vivo. Although the observed replication initiation in the OriA recombinant template is chloroplast-DNAspecific, the mode of replication is different from that observed in vivo with intact ctDNA. However, when the template DNA contains both the OriA and OriB sequences, the in vitro replication proceeds in the theta mode, the mode of replication usually observed in vivo.The complex process of DNA replication has been defined for only a few biological systems such as plasmids, bacteriophages, bacteria and viruses [l-61. So far, little is known about the DNA replication processes in plant systems except for few reports in the field of plant organelles [7-111. Chloroplast DNA (ctDNA) of higher plants seems to be an attractive system for investigation of the molecular details of replication. The chloroplast genomic size of about 150 kb is large but not too large for recombinant manipulations and the genome exists in multiple copies. Understanding the molecular biology of ctDNA replication would not only reveal the detailed characteristics of this system [12-141 but also help solve problems related to stable transformation of the chloroplast organelle. Thus, development of an in vitro replication system derived from higher plant chloroplasts is important both in helping to define DNA metabolism in general as well as advancing the technology of transgenic plant production [15-181. Replicative intermediates of ctDNA from pea leaves have been well characterised using electron microscopic techniques [19]. In pea and maize ctDNA, it was shown that replication begins by forming two displacement loops (Dloops) which expand towards each other to form a theta structure. This small Cairns' structure then proceeds bi-directionally until termination takes place. Meeker et al. [20] have mapped the two replication origins or D-loops by electron Correspondence to S . K.