1. Injection of acetylcholine (ACh, 0.0005-2 micrograms/kg) or glyceryl trinitrate (GTN, 0.01-20 micrograms/kg) into the femoral artery increased femoral artery diameter, femoral blood flow and heart rate, and reduced femoral vascular resistance and systemic arterial blood pressure in anaesthetized dogs. The intravenous (i.v.) injection of ACh (2 micrograms/kg) produced a small decrease in systemic arterial pressure and an increase in heart rate, but did not dilate the hindlimb vessels. 2. Methylene blue, a guanylate cyclase inhibitor, continuously infused into the femoral artery (10 mg/min), attenuated the increase in femoral artery diameter and femoral blood flow, and the decrease in femoral vascular resistance produced by intra-arterial injections of both ACh and GTN. 3. In addition, methylene blue potentiated the decrease in systemic arterial pressure produced by ACh (injected directly into the femoral artery or i.v.), but did not affect the depressor response to GTN. This selective potentiation of ACh-induced hypotension was not affected by autonomic ganglion blockade with hexamethonium (25 mg/kg, i.v.). 4. These results suggest that both ACh- and GTN-induced vasodilatation in vivo occurs through a mechanism involving guanylate cyclase activation in large arteries and resistance vessels in the dog hindlimb. Methylene blue inhibited the local vasodilator actions of ACh in the femoral vasculature despite potentiating the systemic depressor response to that agent.