Technical Abstract: Salivary proteins of Culicoides biting midges are thought to play a key role in the induction of summer eczema (SE), a seasonal recurrent allergic dermatitis in horses. The present study describes the identification of a candidate allergen in artificially collected saliva of the North American species Culicoides sonorensis by immunoblot analysis and mass spectrometry. This candidate was expressed as a polyhistidine-tagged fusion protein in a baculovirus/insect cell expression system, purified using cobalt affinity chromatography, and its clinical relevance was investigated by immunoblotting, histamine release testing (HRT) and intradermal testing (IDT) in eight SE-affected (SE+) and eight control horses. Analysis of native saliva revealed seven proteins ranging from 90 to 15 kDa that were bound by immunoglobulin E (IgE) of horse sera. One immunoreactive protein of 66 kDa (Cul s 1) was further characterized by internal peptide sequencing and identified as maltase, an enzyme involved in sugarmeal digestion. Expressed and purified recombinant Cul s 1 (rCul s 1) was detected by specific IgE in sera of seven SE+ horses whereas only one control animal had rCul s 1-specific IgE. The HRT showed rCul s 1 induced basophil degranulation for all SE+ horses and revealed a considerable higher sensitivity for the recombinant allergen as compared to whole body extracts (WBE) of C. sonorensis. In IDT, rCul s 1 induced immediate, late-phase and delayed skin reactivity in all SE+ horses that had a positive test with Culicoides WBE. None of the control horses showed reactivity with rCul s 1 in HRT and IDT although unspecific reactions were observed for WBE in both test systems. Thus, rCul s 1 is the first described recombinant allergen of Culicoides spp. that may greatly improve in vitro and in vivo allergy diagnosis and may contribute to future immunotherapy of SE in horses.