The purpose of the present study was to identify midline medullary serotonin (5-HT) neurons and to determine if these neurons were distinct from previously identified sympathoinhibitory and sympathoexcitatory neurons. Identification of medullary 5-HT neurons was based on electrophysiological and pharmacological similarities to dorsal raphe 5-HT neurons. Sympathoinhibitory and sympathoexcitatory neurons were characterized by an irregular discharge pattern which was temporally related to inferior cardiac sympathetic nerve discharge (SND) and to the cardiac cycle. Sympathoinhibitory neurons increased their discharge rate and the discharge of sympathoexcitatory neurons decreased during baroreceptor reflex activation. A third type of neuron fired in an extremely regular fashion (as judged by interspike interval analysis), fired at a rate of 1.1 spikes/s and had spike durations of approximately 2 ms. The discharges of regularly firing neurons were not temporally related to SND and were not affected during baroreceptor reflex activation. Regularly firing neurons and sympathoinhibitory neurons could be antidromically activated by electrical stimulation of the intermediolateral cell column of the spinal cord. Axonal conduction velocity of sympathoinhibitory neurons (2.4 m/s) was significantly greater than that for regularly firing neurons (1.3 m/s). Regularly firing neurons were completely inhibited by low doses of the 5-HT1A agonist 8-hydroxy-dipropylamino-tetralin (8-OH-DPAT) (i.e. 2 micrograms/kg, i.v.) while much higher doses of the drug failed to affect the discharges of sympathoinhibitory and sympathoexcitatory neurons. Microiontophoretic application of 5-HT and 8-OH-DPAT profoundly depressed the firing of regularly discharging neurons. Based on the striking similarities between regularly firing medullary neurons and dorsal raphe 5-HT neurons it is concluded that the regularly firing neurons were 5-HT-containing neurons. Furthermore, these medullary 5-HT neurons are distinct from sympathoinhibitory and sympathoexcitatory neurons.