Haemodynamic disturbances leading to ischaemia and reperfusion injury of the digit are thought to be involved in the pathophysiology of acute equine laminitis. Identification of physiological regulators of blood flow through the equine digit is important in identifying factors, which may predispose animals to laminitis. A method was developed to assess endothelium-dependent responses of the isolated Krebs-perfused equine digit by co-administration of 5-hydroxytryptamine (5-HT) with vasodilator agents, carbachol (CCh), bradykinin (BK) and substance P (SP). Bolus co-administration of CCh (0.02–2 μmol), BK and SP (0.02–0.2 nmol), caused inhibition of the 5-HT pressor response by 50–60%. The vasodilator responses were abolished by the detergent, CHAPS, indicating endothelium dependency; whereas vasoconstrictor responses to 5-HT were potentiated. CCh-induced relaxation was significantly reduced by the nitric oxide synthase inhibitor l-NAME (79.7 ± 3.4% inhibition), whereas a large proportion of BK and SP-induced relaxation remained (34.1 ± 6.3% and 33.6 ± 5.3% inhibition). l-NAME potentiated vasoconstrictor responses to 5-HT. In conclusion, this study demonstrates that endothelium-derived NO modulates the response to vasoconstrictors such as 5-HT and is likely to be an important regulator of blood flow in the digital resistance vascular bed. Other factor(s) released by the endothelium are also important in regulating blood flow, whose identity remains to be established.