The spirochaetal bacteria Borrelia burgdorferi (Bb) is the tick-borne causative agent of lyme disease. The major membrane immunogens of Bb are outer surface proteins. The lipid component of these lipoproteins is relevant for the immunogenicity of Bb-lipoproteins. To characterize the antigenic properties, the native lipid component of lipoproteins was isolated and the detailed molecular structure was analyzed. The molecular structure of the lipoprotein–lipid component turned out to be S(propane-2′,-3′diol)-3-thio-2-aminopropanic acid (S-glyceryl-cysteine) with one ester-linked fatty acid, one acetyl group, and one N-terminal amide-bound fatty acid. Fatty acid analysis of the lipid component indicated a heterogeneous composition comprising C16:0, C18:0, C18:1, C18:2, and C 20:0. The antigenicity was tested with in vitro bioassays using human blood-derived dendritic cells (DCs) as antigen-presenting cells and autologous Bb-specific T-cells. We found that human DCs present the lipid component of Bb-lipoproteins via MHC class II inducing an antigen-specific T-cell immune response in vitro.