To determine the functional role of nitric oxide (NO) in regulating vascular conductance during high intensity dynamic exercise in skeletal muscles composed of all major fibre types, female Wistar rats (277 ± 4 g; n = 7) were run on a motor-driven treadmill at a speed and gradient (60 m min-1, 10 % gradient) established to yield maximal oxygen uptake (V˙O2,max). Vascular conductance (ml min-1 (100 g)-1 mmHg-1), defined as blood flow normalised to mean arterial pressure (MAP), was determined using radiolabelled microspheres during exercise before and after NO synthase (NOS) inhibition with N G-nitro-L-arginine methyl ester (L-NAME; 10 mg kg-1, I.A.). The administration of L-NAME increased MAP from pre-L-NAME baseline values, demonstrating that NOS activity is reduced. The administration of L-NAME also reduced vascular conductance in 20 of the 28 individual hindlimb muscles or muscle parts examined during high speed treadmill exercise. These reductions in vascular conductance correlated linearly with the estimated sum of the percentage of slow twitch oxidative (SO) and fast twitch oxidative glycolytic (FOG) types of fibres in each muscle (Δconductance = -0.0082(%SO + %FOG) - 0.0105; r = 0.66; P < 0.001). However, if the reduction in vascular conductance found in the individual hindquarter muscles or muscle parts was expressed as a percentage decrease from the pre-L-NAME value (%Δ = (pre-L-NAME conductance - post-L-NAME conductance)/ pre-L-NAME conductance × 100), then the reduction in vascular conductance was similar in all muscles examined (average %Δ = -23 ± 2 %). These results suggest that NO contributes substantially to the regulation of vascular conductance within and among muscles of the rat hindquarter during high intensity exercise. When expressed in absolute terms, the results suggest that the contribution of NO to the regulation of vascular conductance during high intensity exercise is greater in muscles that possess a high oxidative capacity. In contrast, if results are expressed in relative terms, then the contribution of NO to the regulation of vascular conductance during high intensity exercise is similar across the different locomotor muscles located in the rat hindlimb and independent of the fibre type composition. Experimental Physiology (2001) 86.6, 749-757.