Exercise induced pulmonary haemorrhage (EIPH) is thought to result from capillary stress failure associated with high pulmonary vascular pressures during heavy exercise. One possible mechanism for the caudodorsal distribution of haemorrhage is preferential distribution of blood flow to affected regions due to regional differences in pulmonary vascular regulation. Endothelial cells (EC) are known controllers of vascular smooth muscle tone and could play a role in regional distribution of blood flow. The present study investigated relaxation responses to the endothelium-dependent and independent vasodilators, methacholine (MCh) and nitroglycerin (NTG) in pulmonary arteries from the caudodorsal (top) and cranioventral (bottom) regions of the lung. Pulmonary arteries (7 mm diameter) from 6 horses were isolated from top and bottom regions of the lung and vessels with and without endothelium were mounted in 15 ml tissue baths. Vessels were pre-contracted with norepinephrine (10−6 M) and changes in isometric tension in response to MCh (10−6 M) and NTG (10−6 M) were compared. MCh consistently relaxed, and never contracted, endothelium-intact top vessels. In top vessels, relaxation was abolished by removal of EC, suggesting an obligatory role of EC in MCh relaxation. However, in bottom vessels, a small transient relaxation was followed by a profound contraction, enhanced by EC removal. Relaxation to NTG was similar in all vessels. Therefore, in response to MCh, top vessels relax in an endothelium-dependent manner, while bottom vessels contract. Similar regional differences in endothelium dependent flow mediated vasodilation may result in higher blood flow to the caudodorsal regions of the lung and hence be responsible in part for the location of EIPH.