We have recently shown that nitric oxide (NO) derived from neuronal NO synthase (nNOS) does not contribute to the hyperaemic response within rat hindlimb skeletal muscle during low-speed treadmill running. This may be attributed to low exercise intensities recruiting primarily oxidative muscle and that vascular effects of nNOS-derived NO are manifest principally within glycolytic muscle. We tested the hypothesis that selective nNOS inhibition via S-methyl-l-thiocitrulline (SMTC) would reduce rat hindlimb skeletal muscle blood flow and vascular conductance (VC) during high-speed treadmill running above critical speed (asymptote of the hyperbolic speed versus time-to-exhaustion relationship for high-speed running and an important glycolytic fast-twitch fibre recruitment boundary in the rat) principally within glycolytic fast-twitch muscle. Six rats performed three high-speed treadmill runs to exhaustion to determine critical speed. Subsequently, hindlimb skeletal muscle blood flow (radiolabelled microspheres) and VC (blood flow/mean arterial pressure) were determined during supra-critical speed treadmill running (critical speed + 15%, 52.5 ± 1.3 m min(-1)) before (control) and after selective nNOS inhibition with 0.56 mg kg(-1) SMTC. SMTC reduced total hindlimb skeletal muscle blood flow (control: 241 ± 23, SMTC: 204 ± 13 ml min(-1) (100 g)(-1), P < 0.05) and VC (control: 1.88 ± 0.20, SMTC: 1.48 ± 0.13 ml min(-1) (100 g)(-1) mmHg(-1), P < 0.05) during high-speed running. The relative reductions in blood flow and VC were greater in the highly glycolytic muscles and muscle parts consisting of 100% type IIb+d/x fibres compared to the highly oxidative muscles and muscle parts consisting of 35% type IIb+d/x muscle fibres (P < 0.05). These results extend our understanding of vascular control during exercise by identifying fibre-type-selective peripheral vascular effects of nNOS-derived NO during high-speed treadmill running.