Isoelectric focusing analyses of sera from patients with rheumatoid arthritis (RA) demonstrate two populations of antibodies directed against the Fab portion of pooled human IgG. One population is composed of polyclonal alkaline anti-Fab antibodies (alpha FABA) and the other, acidic alpha FABA which are more clonally restricted. In this study we have identified the immunoglobulin classes and subclasses of these antibodies in RA sera. Enzyme-linked immunosorbent assays (ELISA) demonstrated alpha FABA in RA sera to be predominantly IgG. A large portion of IgG alpha FABA existed as immune complexes, inasmuch as dialysis of RA sera against 6 M urea before ELISA analysis was necessary for maximal detection of alpha FABA activity. Chromatofocusing of RA sera isolated alpha FABA of different charges and revealed the acidic clonally restricted alpha FABA to be IgG4 and IgG3, whereas the polyclonal alkaline group contained IgG1, IgG2, and IgG3. Overall, acidic IgG3 and IgG4 comprised 70% of IgG alpha FABA, and high levels of IgG4 were seen in most RA sera. When alpha FABA were elevated in normal sera, they were primarily of the IgG4 subclass, and also existed as immune complexes. Serum anti-Fab activity was removed by adsorption of sera with Fab fragments. Anti-Fab antibodies of both kappa and lambda light-chain types were present in RA sera, and F(ab')2 fragments of RA serum immunoglobulin were found to possess anti-Fab activity. These studies indicate that alpha FABA in RA sera are limited to the IgG class, and that most of these antibodies exist as immune complexes and display clonal and minor IgG subclass restriction.