Neurons in the rostral ventrolateral medulla (RVLM) were electrophysiologically characterized and anatomically identified using an intracellular recording technique in vivo. Of 49 neurons recorded, 7 were antidromically activated from the dorsolateral funiculus in the thoracic spinal cord, with axonal conduction velocities ranging from 16.6 to 55.0 m/s. The RVLM-spinal neurons were spontaneously active and non-bursting. Additionally, they demonstrated a flat post-R-wave histogram and a flat average of the neuronal membrane potential triggered by the pulsatile arterial pressure. Therefore their activity was not related to cardiac rhythm. Electrical stimulation of the nucleus tractus solitarii (NTS) at the level of the obex evoked monosynaptic excitatory postsynaptic potential (EPSP) on 3 RVLM-spinal neurons; median latency was 1.5 ms. The recorded neurons, intracellularly labeled with horseradish peroxidase (HRP) or biocytin, were located in the rostral pole of the RVLM, between 0.3 and 0.7 mm from the ventral medullary surface and in many cases close to the neurons containing phenylethanolamine-N-methyltransferase (PNMT). These findings are discussed in relation to the physiological role in cardiovascular and nociceptive functional regulation played by the neurons analyzed in this study.