Strongyle parasites are ubiquitous in grazing horses, with cyathostomins being the most prevalent, but the large strongyles having larger clinical impact. Strongylus vulgaris is considered most pathogenic nematode, with migrating larvae causing verminous endarteritis and potentially ischaemic infarction of intestinal segments. Developing anthelmintic resistance in equine parasites has put emphasis on less intensive treatment regimens to maintain efficacy of current anthelmintics. This has been associated with apparent re-emergence of S. vulgaris. Currently there are no methods for diagnosing the pathogenic migrating larval stages of S. vulgaris ante mortem. A cDNA library was constructed from RNA extracted from migrating S. vulgaris larvae. Excretory-secretory antigens from S. vulgaris adult specimens were used to immunise a rat. Hyperimmune serum was used to immunoscreen the cDNA library, an immunoreactive cDNA clone was subcloned into E. coli and the plasmid sequenced, the open reading frame encoding the mature protein was cloned into a pET22b expression vector and expressed as a His-tagged recombinant protein in BL21 expression cells. The recombinant protein was used in an indirect enzyme-linked immunosorbent assay (ELISA) measuring antigen-specific IgG(T) levels. Sera from 22 horses with necropsy-confirmed presence or absence of S. vulgaris were used in the ELISA. The best correlation was seen when analysing antigen-specific IgG(T) levels. Sera from horses with confirmed presence of larval stages of S. vulgaris (n=9) reacted against the recombinant protein, expressed as optic density (OD) readings of >24 % of a positive control, while sera from negative horses had OD readings <2.5 % of the positive control with a sensitivity and specificity of 1. The ELISA shows promise for diagnosing S. vulgaris infection in suspected clinical cases, measuring herd level exposure, and as a potentially useful research tool for investigating possible risk factors associated with development of disease.