Chloroplast DNA from pearl millet (Pennisetum americanum) was used to construct recombinant plasmids. These plasmids contained 97 kilobase pairs of unique DNA sequences. The chloroplast DNA fragments in these plasmids were mapped with the restriction endonucleases SalI, SphI, XhoI, BglI and HpaI. The technique of overlapping hybridization or chromosome walking was used to orient these DNA fragments on a restriction endonuclease map of the chloroplast genome. The size of the chloroplast DNA from pearl millet was estimated in this fashion to be 127–138 kilobase pairs. Twenty one kilobase pairs of the cloned DNA fragments were represented twice on the genome as inverted repeats. Thus, the recombinant plasmids which were isolated contained approximately 86–93% of the nucleotide sequences in the chloroplast genome of pearl millet. Previously characterized cloned chloroplast DNA sequences from other plants were used as hybridization probes to locate the genes for the large subunit of ribulose 1,5-bisphosphate carboxylase, the β-coupling factor of ATPase and the 32 kilodalton polypeptide of photo system 11 on the restriction endonuclease map of the pearl millet chloroplast genome.