The Rickettsia prowazekii citrate synthase (gltA) gene, previously cloned in Escherichia coli, was localized to a 2.0-kilobase chromosomal fragment. DNA sequence analysis of a portion of this fragment revealed an open reading frame of 1,308 base pairs that encodes a protein of 435 amino acids with a molecular weight of 49,171. This translation product is comparable in size to both the E. coli and pig heart citrate synthase monomers and to the protein synthesized in E. coli minicells containing the rickettsial gene. Comparisons between the deduced amino acid sequence of R. prowazekii citrate synthase and those of the E. coli and pig heart enzymes revealed extensive homology (59%) between the two bacterial proteins. In contrast, only 20% of the rickettsial enzyme residues were shared with the functionally similar pig heart enzyme residues. Upstream from the open reading frame and in close proximity to one another, sequences with homology to E. coli consensus sequences for RNA polymerase and ribosome binding were identified. S1 nuclease mapping experiments demonstrated that the start of transcription for this gene in E. coli was located in the upstream region. Codon usage in the rickettsial gltA gene was found to be very biased and differed from the pattern observed in E. coli. Adenine and uracil were used preferentially in the third base position of rickettsial codons.