Advanced peripheral vascular disease, critical limb ischaemia (CLI) is associated with an inability to regulate blood volume within the capillary beds of the diseased part of the leg. Part of the sequelae accompanying CLI can be associated with dysfunction of resistance arterioles. Reactive oxygen species (ROS) were originally considered toxic metabolites. Increasing evidence suggests that they can play an important signalling role in essential physiological functions including pressure-dependent arteriolar myogenic tone (1). In addition to the well documented calcium/ calmodulin/ myosin light chain kinase dependency of myogenic contraction evidence suggests that G and F actin based structures contribute to myogenic tone (2). Here ROS and G/F actin mechanisms of arteriolar contractile responses in skeletal muscle arterioles isolated from the diseased part of the leg (DSM) vs. arterioles isolated form the healthy part of the leg (PSM, internal control) have been examined. Methods: Resistance arterioles (lumen diameter ~ 80μM) were isolated from skeletal muscle biopsies taken from subjects with CLI. Pressure-dependent and pressure-independent mechanisms of vascular tone were studied (Danish MyoTech P110 pressure myograph). Results: There was no difference in pressure-independent mechanisms of vascular tone (DSM vs. PSM). Pressure-dependent myogenic constriction was reduced (20.9 ± 2.3% vs. 4.2 ± 1.2% and 28.2 ± 3.1% vs. 6.8 ± 1.4%, PSM vs. DSM at 80mmHg and 120mmHg respectively; n=6 pairs). The antioxidant NAC, the inhibitor of NAD(P)H oxidase DPI and cytochalasin D, an inhibitor of actin polymerisation inhibited myogenic responses in PSM arterioles. NAC, DPI and cytochalasin D had no effect on pressure-dependent myogenic contraction measured in the DSM arterioles. NAC, DPI and cytochalasin D actions were selective to myogenic contraction as they had no effect on pressure-independent contraction. Discussion: Based on these results we confirm that the myogenic response involves two discrete contractile components2. One pathway involves the conventional signalling pathway for vascular smooth muscle constriction. The other involves activation of NAD(P)H oxidase, elevation of ROS and actin polymerization. Since the NAD(P)H/ROS/actin polymerization-dependent response is absent in arterioles associated with CLI, we propose that deregulation of this second pathway negates the ability of these arterioles to resist pressure-dependent forced dilatation and generate a myogenic contraction and this dysfunction significantly contributes to the altered vascular function associated with CLI.