The genes encoding three enzymes of the glycolytic pathway have been identified and sequenced completely in Borrelia burgdorferi sensu stricto and partially in B. hermsii. They are clustered on the chromosome into an operon with a single putative promoter and are arranged downstream of this promoter in the following order: gapdh (glyceraldehyde-3-phosphate dehydrogenase), pgk (phosphoglycerate kinase), and tpi (triosephosphate isomerase). gapdh and pgk are separated by 19 bp of intergenic sequence and pgk and tpi are separated by only 1 bp. Each of the three genes contains a putative RBS 6-7 bp upstream of each respective translational (ATG) start codon. The deduced protein encoded by gapdh consists of 335 amino acids (aa) with a predicted MW of 36,400, that of pgk is 393 aa (MW of 42,156) and that of tpi is 290 aa (MW of 27,683). The aa sequences of each of the three enzymes share 58.4% (GAPDH), 52.8% (PGK) and 46.1% (TPI) identity with respective enzymes from other prokaryotic organisms. Phylogenetic analyses based on these universal and conserved proteins support the hypothesis that spirochetes are an ancient and distinct eubacterial phylum.