In order to identify and characterize the wheat grain allergens involved in bakers' asthma, proteins were sequentially extracted from whole-meal flour. The polypeptide composition of the individual solubility fractions (albumin/globulin, gliadin and glutenin) was analyzed by sodium dodecyl sulfate-gel electrophoresis (SDS-PAGE), and high-resolution two-dimensional gel electrophoresis with immobilized pH gradient 4–9 in the first dimension (IPG-Dalt). The resolved polypeptides were transferred onto an immobilizing polyvinylidene difluoride membrane and incubated with a pooled serum from four asthmatic bakers. Bound IgE was demonstrated by autoradiography using 125I-labeled anti-human IgE. Our study demonstrated that the serum of the bakers allergic to flour contained IgE antibodies which bound to numerous polypeptides of all three solubility fractions. The highest percentage of IgE binding was observed with certain albumin and/or globulin polypeptides, whereas the gliadins and glutenins exhibited considerably less allergenicity. SDS-PAGE revealed that the protein which bound the highest percentage of IgE from the sera of the allergic bakers is a 27 kDa albumin. More detailed investigations using IPG-Dalt demonstrated that this allergen is not a single polypeptide but consists of several polypeptide spots that differ in their isoelectric points. Quantitative studies using computer-assisted laser densitometry revealed that the amount of patients' IgE bound by these particular polypeptides differed considerably between the seven wheat cultivars examined, ranging from 13% to 53% of the total radioactive uptake.