Leptospiral LPS plays a critical role in immunity to leptospirosis and forms the basis for serological classification of Leptospira. However, neither the structure of leptospiral LPS nor the genetics of its biosynthesis have been elucidated. A probe derived from the rhamnose biosynthetic genes of L. interrogans serovar Copenhageni was used to identify the rfb locus of L. borgpetersenii serovar Hardjobovis. Chromosome walking and sequence analysis revealed an rfb locus spanning 36.7 kb, which consists of 31 ORFs transcribed in the same direction. Clusters of genes were identified which encode proteins related to enzymes involved in the biosynthesis of activated sugars including rhamnose. Additional ORFs in the locus encode glycosyltransferases for the assembly of the O-antigen subunit and integral membrane proteins for the transport of O-antigen subunits through the membrane and assembly into LPS.