Assays for specific antigen-binding activity were performed on sera from 172 patients with monoclonal macroglobulinemia defined by immunofixation electrophoresis. The sera were collected between 1970 and 2002. Mean IgM level was 1,409 mg/dL with a range from 70 to 6,800. Cryoglobulins were identified in 15.3% (26/170 sera: 12 trace, five single component, and nine mixed IgM-IgG). Rheumatoid factor (RF) was detected in 19 of 151 (12.6%) samples with titers ranging from 1:80 to 1:327,680. Among the nine mixed IgM-IgG cryos, eight were RF-positive and six of six displayed positivity for hepatitis C virus. Cold agglutinins (CA) were present in 8.5% (10/117) of sera with anti-I titers between 1:512 and 1:65,536. IgM binding to a series of glycosaminoglycan oligosaccharides, glycolipids, and glycoprotein antigens was found in 75 samples (43%). IgM binding to antigens having known associations to polyneuropathies occurred in 20 patients (12%). Anti-nuclear antibody (ANA) was documented in 10.7% (18/ 169) of sera. Anti-DNA activity was absent in all samples tested. Sera from 71% of patients with monoclonal macroglobulinemia in this series exhibited binding to autoantigens. Some of these immune complexes resulted in clinically significant manifestations. Our results suggest that many monoclonal immunoglobulins may be functional antibodies rather than "paraproteins." Characterization of antigen-binding activities may provide insight into the pathogenesis of monoclonal gammopathies.