Presynaptic blockade of cutaneous vasoconstrictor nerves (VCN) abolishes the axon reflex (AR) during slow local heating (SLH) and reduces the vasodilator response. In a two-part study, forearm sites were instrumented with microdialysis fibers, local heaters and laser-Doppler flow probes. Sites were locally heated from 33 to 40°C over 70 min. In Part 1, we tested whether this effect of VCN acted via nitric oxide synthase (NOS). In 5 subjects, treatments were: (a) untreated, (b) bretylium, preventing neurotransmitter release, (c) L-NAME to inhibit NOS, and (d) combined bretylium+L-NAME. At treated sites the AR was absent and there was an attenuation of the ultimate vasodilation (P<0.05), which was not different among those sites (P>0.05). In Part 2, we tested whether norepinephrine (NE) and/or neuropeptide Y (NPY) is involved in the cutaneous vasodilator response to SLH. In 7 subjects, treatments were: (a) untreated, (b) propranolol and yohimbine to antagonize - and -receptors, (c) BIBP3226 to antagonize Y₁-receptors, and (d) combined propranolol+yohimbine+BIBP3226. Treatment with propranolol+yohimbine or BIBP3226 significantly increased the temperature at which AR occurred (n=4) or abolished it (n=3). The combination treatment consistently eliminated it. Importantly, ultimate vasodilation with SLH at the treated sites was significantly (P<0.05) less than at the control. These data suggest that NE and NPY are important in the initiation of the AR and for achieving a complete vasodilator response. Since VCN and NOS blockade in combination do not have an inhibition greater than either alone, these data suggest that VCN promote heat-induced vasodilation via an NO-dependent mechanism.