To investigate the relative importance of inhaled particulates and soluble components in the response to inhaled hay dust suspension (HDS), heaves (previously termed chronic obstructive pulmonary disease; n = 7) and control (n = 6) horses were given inhalation challenges with whole and fractionated HDS. Fractionation was achieved by centrifugation to yield supernatant (SUP) and particulate debris. The particulates were then washed repeatedly in saline to produce a washed particulate (WP) fraction which comprised mainly fungal spores, and a wash fraction (WF) which comprised saline and soluble components washed from the surface of the particulates. Inhalation of HDS and SUP induced a significant airway neutrophilia in both groups, with the magnitude of the response being significantly greater in heaves horses. SUP induced significantly less airway neutrophilia than HDS in both groups, despite the endotoxin and protease content of HDS and SUP being comparable. WP and WF induced only a slight airway neutrophilia in heaves horses. However, a combined challenge with SUP and WP induced a neutrophilic response approaching the magnitude of that following HDS challenge, indicating that dust particulates contribute to the pulmonary recruitment of neutrophils in heaves. Consequently, inhalation challenge with HDS, which contains both particulates and soluble dust components, may be a more useful tool for the diagnosis and investigation of heaves than aqueous dust extracts, which contain only soluble components.