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 [third thoracic (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-hydroxytryptamine (HT)(2A) antagonist trans-4-((3Z)3-[(2-dimethylaminoethyl)oxyimino]-3-(2-fluorophenyl)propen-1-yl)-phenol, hemifumarate (SR-46349B, 80 microg/kg in 0.8 ml) to the cerebrospinal fluid 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 micromol in 0.5 ml) 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.