Allergic bronchopulmonary aspergillosis (ABPA), a severe pulmonary complication caused by Aspergillus fumigatus, is considered a complex clinical syndrome with defined serological, pathological radiological and clinical features. The diagnosis of ABPA is often difficult because of several overlapping clinical and laboratory findings shared between asthma with sensitization to A. fumigatus and ABPA, but essential for treatment to prevent severe deterioration of pulmonary function. We have cloned A. fumigatus allergens from a cDNA library displayed on phage surface, sequenced the inserts and produced recombinant proteins in Escherichia coli. The single recombinant allergens were used to assess the immunological response in representative groups of A. fumigatus-sensitized asthmatics with or without ABPA and healthy controls. The allergens rAsp f 1, a 16.9 kDa ribotoxin, rAsp f 3, a 18.5 kDa peroxisomal protein, and rAsp f 6, a 23 kDa manganese superoxide dismutase, were identified as proteins with known biological function and rAsp f 4, a 30 kDa allergen, lacks sequence homology to known proteins. The secreted ribotoxin rAsp f 1 and rAsp f 3 are recognized by serum IgE of A. fumigatus-sensitized asthmatics with or without ABPA, whereas the non-secreted manganese superoxide dismutase rAsp f 6 and the rAsp f 4 allergen are exclusively recognized by serum IgE of ABPA patients. The dissection of IgE-mediated immune responses to single recombinant A. fumigatus allergens in asthmatic patients allow a discrimination between ABPA and A. fumigatus sensitization with high specificity (100%) and sensitivity (90%).