Chronic inflammation associated with osteoarthritis (OA) may alter normal vascular responses and contribute to joint degradation. Vascular responses to vasoactive mediators were evaluated in the ACL deficient knee. Chronic joint instability and progressive OA were induced in rabbit knees by surgical transection of the ACL. Under halothane anaesthesia, laser speckle perfusion imaging (LSPI) was used to measure medial collateral ligament (MCL) blood flow in unoperated control (n=12) and 6-week ACL-transected knees (n=12). Acetylcholine (Ach), bradykinin, histamine, substance P (SP), and prostaglandin E₂ (PGE2) were applied to the MCL vasculature in topical boluses of 100µl (dose range 10^-14 to 10^-8mol). In normal joints, Ach, bradykinin, histamine and PGE2 evoked a dilatory response. Substance P caused a biphasic response which was dilatory from 10^-14 to 10^-11 moles and constricting at higher doses. In ACLdeficient knees, ACh, bradykinin, histamine and SP decreased perfusion while PGE₂ had a biphasic response which decreased perfusion at 10^-14 moles to 10^-11 moles, and was dilatory at higher concentrations. Sodium nitroprusside increased perfusion in resting and phenylephrine pre-contracted vessels with no significant differences between ACL transected and control knees. Femoral artery occlusion and release increased perfusion by 74.3 + 11.1 % in control knees, but only by 25.8 + 4.4 % in ACL-deficient knees. The altered responsiveness of the MCL vasculature to these inflammatory mediators may indicate endothelial dysfunction in the MCL, which may contribute to the progression and severity of OA, and to the adaptation of the joint in an altered mechanical environment.