Preparative thin-layer chromatograms of chloroform-methanol extracts of Borrelia burgdorferi (B31) sonicates showed four fractions (Rf values of 0.84, 0.81, 0.66 and 0.61) that stained with iodine vapors, orcinol, or phospray, suggesting the presence of lipid-, carbohydrate-, and phosphorus-containing compounds. Sera from patients with Lyme disease showed IgM or IgG antibody reactivity to hydrophobic fractions, designated F1 and F2, in both early and late stages of the disease. Lack of constitutive amino acids in these fractions was shown by protein, amino acid, and peptide detection analyses. Sera from patients with syphilis, systemic lupus erythematosus, and antiphospholipid syndrome reacted to one or both of the fractions. Adsorption of sera from Lyme disease patients with intact B. burgdorferi resulted in significantly different pre- and postadsorption patterns of reactivity by whole cell ELISA, whereas adsorption with F1 and F2 resulted in similar pre- and postadsorption patterns. These fractions may not be present in aqueous whole cell or whole cell lysate ELISA antigens or in immunoblots.