Lyme disease (LD) is a tick-transmitted bacterial infection caused by Borreliella burgdorferi and other closely related species collectively referred to as the LD spirochetes. The LD spirochetes encode an uncharacterized family of proteins originally designated rotein amily welve (PF12). In B. burgdorferi strain B31, PF12 consists of four plasmid-carried genes, encoding BBK01, BBG01, BBH37, and BBJ08. Henceforth, we designate the PF12 proteins amily welve ipoprotein (Ftl) (FtlA) (BBK01), FtlB (BBG01), FtlC (BBH37), and FtlD (BBJ08). The goal of this study was to assess the potential utility of the Ftl proteins in subunit vaccine development. Immunoblot analyses of LD spirochete cell lysates demonstrated that one or more of the Ftl proteins are produced by most LD isolates during cultivation. The Ftl proteins were verified to be membrane associated, and nondenaturing PAGE revealed that FtlA, FtlB, and FtlD formed dimers, while FtlC formed hexamers. Analysis of serum samples from B. burgdorferi antibody (Ab)-positive client-owned dogs ( = 50) and horses ( = 90) revealed that a majority were anti-Ftl Ab positive. Abs to the Ftl proteins were detected in serum samples from laboratory-infected dogs out to 497 days postinfection. Anti-FtlA and FtlB antisera displayed potent complement-dependent Ab-mediated killing activity, and epitope localization revealed that the bactericidal epitopes reside within the N-terminal domain of the Ftl proteins. This study suggests that FtlA and FtlB are potential candidates for inclusion in a multivalent vaccine for LD.