A histamine inhalation challenge (HIC) procedure was developed to assess hyperreactive states in horses. Following clinical evaluation, percutaneous lung biopsies were performed on nine light breed mares aged 6 to 15 years. Five horses, with normal small airways, were classified as group A and four subjects with small airway disease (SAD) lesions formed group B. Pulmonary mechanics parameters were monitored following an aerosol of 0.9% saline and every 5 min for up to 30 min after HIC with 0.5% w/v of histamine diphosphate, administered through a face mask for 2.5 min. Tidal volume (VT) and airflow (V) values were obtained with a pneumotachograph. Transpulmonary pressure (delta Ppl) was measured by the esophageal balloon catheter method. Dynamic compliance (Cdyn), total pulmonary resistance (RL), end expiratory work of breathing (EEW) and respiratory rate (f) were calculated by a pulmonary mechanics computer. Group A horses had increases in RL, and decreases in Cdyn whereas horses in group B were hyperreactive and showed greater changes in EEW, Cdyn, and delta Ppl but with a relatively lower variation of RL. One horse in clinical remission from SAD, but with a high biopsy score (group B), and one clinically normal horse belonging to group A showed marked hyperreactivity as shown by increases in EEW, maximum change in delta Ppl and RL and decreases in Cdyn. These results suggest that the HIC described can be used as a method to investigate airway hyperreactivity and SAD in horses.