Several studies have demonstrated that proteins homologous to the Alt a 1 major allergen of Alternaria alternata are expressed in other members of the Pleosporaceae family. However, since no direct biochemical data have been reported concerning the presence of Alt a 1 allergen homologues capable of binding IgE in the excretion-secretion products of Stemphylium and Ulocladium, our objective was to explore their presence in Stemphylium botryosum and Ulocladium botrytis. S. botryosum and U. botrytis culture filtrate extracts were analyzed by two-dimensional (2D)-electrophoresis and 2D-immunoblotting using polyclonal rabbit antibodies raised against recombinant Alt a 1, as well as five human sera from patients allergic to Alternaria. Cross-reactivity immunoassays were performed by ImmunoCAP inhibition and 2D-immunoblotting inhibition. IgE-binding proteins recognized by the rabbit antiserum raised against Alt a 1, with apparent molecular weights of 17-18 kDa and isoelectric points of 4, were identified as Alt a 1-like proteins. Alt a 1 inhibited IgE-specific binding to the Alt a 1 homologues from S. botryosum and U. botrytis. In conclusion, it was demonstrated that allergens which are homologous to Alt a 1 are expressed in the excretory-secretory materials of the phylogenetically-related species S. botryosum and U. botrytis.