Sympathetic-derived NPY helps regulate inflammatory responses in injury and disease, is a vasoconstrictor and stimulates angiogenesis. Rupture of the anterior cruciate ligament (ACL) is a common clinical presentation that results in tissue inflammation, hyperaemia and angiogenesis in the intact medial collateral ligament (MCL). This study is the first to examine the vasoregulatory role of NPY in ACL deficient knee joints using the newly developed technique of laser speckle perfusion imaging (LSPI). MCL blood flow was measured in two groups of adult rabbits: unoperated control (n=6), and 6-week ACL-transected (n=5). Under anaesthesia, the MCL was surgically exposed and tissue blood flow was imaged at high resolution using LSPI. NPY was applied to the MCL vasculature in topical boluses of 100µl (dose range 10^-14 to 10^-9mol), and the alpha-adrenoceptor agonist phenylephrine was applied in doses of 10^-14, 10^-10 and 10^-7 mol. In control rabbits, topical administration of NPY or phenylephrine produced dose-dependent vasopressor responses (maximal effect at 10^-9 mol NPY and 10^-7mol phenylephrine). In ACL-transected knees there was little or no vasoconstrictive response to NPY at any dose. The response to phenylephrine was significantly reduce in comparison to control ligaments. Possible causes of the reduced vasoconstrictive response to NPY in the MCL after 6wk of ACL-deficiency include: development of tolerance to the peptide due to a prolonged increase in sympathetic nerve activity or change in the distribution or functionality of the NPY Y1 receptors. Chronic ACL deficiency leads to profound and protracted hyperaemia in associated articular tissues. Abrogation of a vasoconstrictor response to both NPY and phenylephrine in the MCL indicates that ACL deficiency induces major changes in the vascular physiologic homeostasis.