A 5.6-kbp fragment of Spiroplasma citri DNA containing the dnaA gene has been cloned and sequenced. Nucleotide sequence analysis shows that this fragment harbors the genes for the replication initiator protein (dnaA), the beta subunit of DNA polymerase III (dnaN), and the DNA gyrase subunits A and B (gyrA and gyrB). The arrangement of these genes, dnaA-dnaN-gyrB-gyrA, is similar to that found in all Gram-positive bacterial genomes studied so far, except that no recF gene was found between dnaN and gyrB. Several DnaA-box consensus sequences were found upstream of dnaA and in the dnaA-dnaN intergenic region. The dnaA region with the flanking DnaA-boxes and the tetracycline resistance determinant, tetM, were linked into a circular recombinant DNA. This DNA was able to replicate autonomously when introduced by electroporation into S. citri cells. These experiments show that the dnaA region with the DnaA-boxes is the origin of replication of S. citri and can be used to construct gene vectors.