Sera from patients with primary syphilis have been fractionated by numerous investigators in order to explore the serological reactivity of the various classes of immunoglobulin. The methods applied have been the following: Electrophoresis (Laurell, 1955; Laurell and Lindau, 1958; Portnoy, Julian, Smith and Harris, 1963); Density gradient ultracentrifugation (Aho, 1967; Davis, Moore, Kabat, and Harris, 1945); DEAE cellulose column chromatography (Laurell and Malmquist, 1961); Gel filtration on Sephadex G 200 column (Aho, 1967; Julian, Logan, and Norins, 1969; Kiraly and Jobbagy, 1969). In the present work sera from patients with primary syphilis were gel-filtered and distributed into ten fractions in order to obtain detailed resolution. All active serum fractions were tested with the rapid plasma reagin (RPR) test, complement-fixation reactions with cardiolipin antigen (Card. C'FR) and T. reiteri protein antigen (RPC'FR), the fluorescent treponemal antibody (FTA) test, the fluorescent treponemal antibody test with sera absorbed by T. reiteri suspension (FTA-ABS), and the T. pallidum immobilization (TPI) test in order to examine the serological reactivity of immunoglobulin classes IgM and Ig G in the presence of heat-labile interfering substances. It is well known that, of the various serological reactions, the TPI is the last to become positive. Thus TPI non-reactive sera were regarded as representating a shorter duration of infection than TPI reactive sera. The aim was to obtain information about the sequence of antibody formation in humans by examining the serological reactivity of the different classes of immunoglobulin. In addition, sera from three patients were fractionated after treatment of syphilis to ascertain the sequence in which the serological reactions of the immunoglobulin classes reversed to negative.