Neurons in the rostral medullary raphe parapyramidal region regulate cutaneous sympathetic nerve discharge. Using focal electrical stimulation at different dorsoventral raphe/parapyramidal sites in anesthetized rabbits, we have now demonstrated that increases in ear pinna cutaneous sympathetic nerve discharge can be elicited only from sites within 1 mm of the ventral surface of the medulla. By comparing the latency to sympathetic discharge following stimulation at the ventral raphe site with the corresponding latency following stimulation of the spinal cord (T3 dorsolateral funiculus) we determined that the axonal conduction velocity of raphe-spinal neurons exciting ear pinna sympathetic vasomotor nerves is 0.8±0.1 m/s (n=6, range 0.6-1.1 m/s). Applications of the 5-HT2A antagonist, trans-4-((3Z)3-[(2-Dimethylaminoethyl)oxyimino]-3-(2-fluorophenyl)propen-1-yl)-phenol, hemifumarate (SR 46349B, 80 µg/kg in 0.8 ml) to the CSF above thoracic spinal cord (T1-T7), but not the lumbar spinal cord (L2-L4), reduced raphe-evoked increases in ear pinna sympathetic vasomotor discharge from 43±9 to 16±6% (P<0.01, n=8). Subsequent application of the excitatory amino acid (EAA) antagonist kynurenic acid (25 µmoles in 0.5ml) substantially reduced the remaining evoked discharge (22±8 to 6±6%, P<0.05, n=5). Our conduction velocity data demonstrate that only slowly conducting raphe-spinal axons, in the unmyelinated range, contribute to sympathetic cutaneous vasomotor discharge evoked by electrical stimulation of the medullary raphe/parapyramidal region. Our pharmacological data provide evidence that raphe-spinal neurons using 5-HT as a neurotransmitter contribute to excitation of sympathetic preganglionic neurons regulating cutaneous vasomotor discharge. Raphe-spinal neurons using an EAA, perhaps glutamate, make a substantial contribution to the ear sympathetic nerve discharge evoked by raphe stimulation.