The immunochemical properties of antigens produced by Aspergillus fumigatus were investigated with biochemical purification techniques in conjunction with the production of murine monoclonal antibodies (MAbs) and binding studies with human IgG and IgE antibodies. A. fumigatus antigens were partially purified by gel filtration and hydrophobic interaction chromatography on phenyl-Sepharose. Two fractions that eluted with either 2 mol/L or 0.15 mol/L of NaCl demonstrated strong binding to human IgG and IgE antibodies. Immunoprecipitation analysis with IgG antibodies from six patients with different Aspergillus-related diseases demonstrated that the 2M and 0.15M fractions contained major antigens of molecular weight 18 kd (Asp f I) and 45 kd, respectively. The 125I-labeled 2M fraction was used to compare IgG antibodies to A. fumigatus in sera from 25 patients with Aspergillus-related diseases. IgG antibodies were significantly higher in patients with allergic bronchopulmonary aspergillosis (geometric mean, 437 U/ml) than in patients with asthma (geometric mean, 14 U/ml; p less than 0.001), but undetectable (less than 5 U/ml) in 43/48 control subjects. A good correlation was found between levels of IgG antibodies to the 125I-labeled 0.15M fraction and the 125I-labeled 2M fraction in sera from 106 patients with cystic fibrosis (r = 0.77; p less than 0.001). Five murine IgG MAbs and two IgM MAbs were raised against the 2M fraction, and immunoprecipitation with the IgG MAb demonstrated two distinct antigens within the 2M fraction, Asp f I, and a 16 kd antigen. The results of a solid-phase RIA with IgG MAb 4A6 demonstrated that approximately 85% of A. fumigatus-allergic patients with allergic bonchopulmonary aspergillosis had IgE antibodies to Asp f I. The three protein antigens defined in these studies are useful probes for investigating the immunopathogenesis of diseases associated with colonization by A. fumigatus.