Horses frequently suffer from respiratory diseases, which irrespective of aetiology, are often associated with airway mucus accumulation. Studies on human airways have shown that the key structural components of the mucus layer are oligomeric mucins, which can undergo changes of expression and properties in disease. However, there is little information on these gel-forming glycoproteins in horse airways mucus. Therefore, the aims of this study were to isolate equine airways oligomeric mucins, characterize their macromolecular properties and identify their gene products. To this end, pooled tracheal washes, collected from healthy horses and horses suffering from respiratory diseases, were solubilized with 6M guanidinium chloride. The oligomeric mucins were purified by density gradient centrifugation followed by size exclusion chromatography. Biochemical and biophysical analyses showed the mucins were stiffened random coils in solution that were polydisperse in size (M_r 6-20 MDa, average M_r 14 MDa) and comprised of disulfide-linked subunits (average M_r 7 MDa). Agarose gel electrophoresis showed that the pooled mucus sample contained at least two populations of oligomeric mucins. Electrospray ionization tandem mass spectrometry of tryptic digests of the unfractionated mucin preparation showed that the oligomeric mucins Muc5b and Muc5ac were present. In summary, we have shown that equine airways mucus is a mixture of Muc5b and Muc5ac mucins which have a similar macromolecular organization to their human counterparts. This study will form the basis for future studies to analyze the contribution of these two mucins to equine airways pathology associated with mucus accumulation.