The structure and composition of periplasmic flagella (PF) of Leptospira interrogans were analyzed. Electron microscopic observations showed that leptospiral PFs are complex structures composed of an 11.3 nm diameter core and two sheaths. Image analysis revealed sheathed PFs with two different diameters; one of 21.5 nm diameter and the other 42 nm diameter. Two dimensional protein electrophoresis of purified PFs demonstrated the presence of at least seven proteins. The molecular masses of the PF proteins ranged from 31.5 kDa to 36 kDa. Monoclonal antibodies against PF proteins and polyclonal antibodies against gel-purified PF proteins were developed. Immunoelectron microscopy studies demonstrated that a 36 kDa protein was associated with one of the PF sheaths (21.5 nm diameter filament) whereas two antigenically related proteins, the 34 kDa and 35.5 kDa, are located in the core of the leptospiral PF. The 34 kDa and the 35.5 kDa proteins had similar isoelectric point and their N-termini had amino acid sequence homology to PF core proteins found in other spirochetes. These two proteins were named FlaB1 and FlaB2, respectively. Monoclonal antibodies against a major 31.5 kDa PF proteins (MoAb 4A9) did not bind a visible PF structure. A DNA library of L. borgpetersenii DNA was constructed using gt11 as the vector. The library was screened with a pool of monoclonal antibodies against L. interrogans PF. Fifteen reactive plaques were selected. All the clones appeared to react with MoAb 4A9, which recognizes at least 5 L. borgpetersenii PF proteins. Southern blot analysis and DNA sequence analysis separated the clones into 4 groups. One of the clones, expressing the largest fusion protein, was chosen for further analysis. Nucleotide sequences flanking the original clone were obtained and sequenced. An open reading frame (ORF) of 604 bases was observed. The amino acid sequence derived from the DNA analysis showed similarity to an Escherichia coli signal peptidase SppA and to an uncharacterized ORF found near the Haemophilus influenzae lic-1 locus.