The structure and composition of periplasmic flagella (PF) from Leptospira interrogans serovar pomona type kennewicki were characterized. Electron microscopic observations showed that leptospiral PF were complex structures composed of an 11.3-nm-diameter core surrounded by two sheath layers with 21.5- and 42-nm diameters. Two-dimensional gel electrophoresis of isolated PF showed the presence of seven different proteins ranging in mass from 31.5 to 36 kDa. Rabbit polyclonal and mouse monoclonal antibodies against PF proteins were prepared and were used to localize specific proteins to portions of the PF structure by immunoelectron microscopy. A 34-kDa protein was associated with the 11.3-nm-diameter core filament, while a 36-kDa protein was associated with a PF sheath (21.5-nm-diameter filament). The amino termini of the 34- and 35.5-kDa proteins were homologous to PF core proteins of other spirochetes. The experimental data suggested that L. interrogans PF contains 2 proteins (34 and 35.5 kDa) in the PF core.