The immunoglobulin (Ig) class and IgG subclasses of autoantibodies to commercial VDRL antigen, creatine kinase (CK), and fibronectin (Fn) in the sera of patients with various stages of syphilis were quantified using solid-phase radioimmunoassays (RIA) and ELISA. Sera from patients with active secondary syphilis, initially positive for anti-Fn and anti-CK autoantibodies by RIA, were re-evaluated by ELISA using monoclonal antibodies (MoAb) for detection of human Ig class and subclass responses. Results of these assays revealed that anti-Fn and anti-CK responses were not only IgG in nature, but dramatically skewed to IgG1 and IgG3 subclasses. While the restricted, co-expression of these isotypes seemingly paralleled anti-treponemal activity, inverse relationships actually existed between the subclass responses to Fn and those to Treponema pallidum. In contrast, anti-VDRL were predominantly IgM in 17 of 22 patients. Of those sera exhibiting detectable anti-VDRL IgG activity, responses appeared to be restricted to IgG1. These results suggest that different control mechanisms may be responsible for regulation of the various autoantibody responses expressed during syphilitic infection.