We tested the hypothesis that ACh or nitric oxide (NO) might be involved in the vasodilation that accompanies a single contraction of the forearm. Eight adults (3 women and 5 men) completed single 1-s-duration contractions of the forearm to raise and lower a weight equivalent to approximately 20% maximal voluntary contraction through a distance of 5 cm. In a second protocol, each subject had a cuff, placed completely about the forearm, inflated to 120 mmHg for a 1-s period, then released as a simulation of the mechanical effect of muscle contraction. Three conditions were studied, always in this order: 1) control, with intra-arterial infusion of saline; 2) after muscarinic blockade with atropine; and 3) after NO synthase inhibition with NG-monomethyl-L-arginine (L-NMMA) plus atropine. Forearm blood flow (FBF), measured by combined pulsed and echo Doppler ultrasound, was reduced at rest with L-NMMA-atropine compared with the other two conditions. After the single contraction, there were no effects of atropine, but L-NMMA reduced the peak FBF and the total postcontraction hyperemia. After the single cuff inflation, atropine had no effects, whereas L-NMMA caused changes similar to those seen after contraction, reducing the peak FBF and the total hyperemia. The observation that L-NMMA reduced FBF in response to both cuff inflation and a brief contraction indicates that NO from the vascular endothelium might modulate the basal level of vascular tone and the mechanical component of the hyperemia with exercise. It is unlikely that ACh and NO from the endothelium are involved in the dilator response to a single muscle contraction.