Inhalation challenges with antigenic extracts of Micropolyspora faeni and Aspergillus fumigatus induced pulmonary disease, similar to naturally occurring chronic obstructive pulmonary disease (COPD), only in COPD affected horses, implicating these agents in the aetiology of equine COPD. In this study, bronchoalveolar lavage fluid (BALF) cytology examination proved to be the most valuable technique for detecting pulmonary disease. Two methods for standardising the variable recovery of pulmonary epithelial lining fluid (PELF), in BALF, namely the urea and albumen dilution techniques, were evaluated in the horse. While both techniques proved to be satisfactory, the former was considered to be more accurate. Comparison of the cellular and molecular components of BALF collected from different lung segments suggested that the composition of PELF is uniform throughout the lungs of control and symptomatic COPD affected horses. COPD affected horses had a significantly increased PELF histamine concentration at 5h, but not at 0.5h, after 'natural (hay and straw) challenges', suggesting involvement of a late phase, pulmonary mast cell/basophil mediated hypersensitivity response in the pathogenesis of equine COPD. Natural challenge significantly increased the ratios of CD4+ , T helper/inducer lymphocytes and significantly reduced the ratios of CD8+ , T suppressor/cytotoxic lymphocytes in BALF from COPD affected horses, suggesting that T lymphocytes have an important role in the pathogenesis of equine COPD. Prior to challenge, COPD affected horses had significantly higher BALF B lymphocyte ratios than controls, suggesting a role for B lymphocytes in the pathogenesis of equine COPD.