--- - |2+ Key points To maintain appropriate blood flow to various tissues of the body under a variety of physiological states, autonomic nervous system reflexes regulate regional sympathetic nerve activity and arterial blood pressure. Our data obtained in anaesthetized rats revealed that glycine released in the rostral ventrolateral medulla (RVLM) plays a critical role in maintaining arterial baroreflex sympathoinhibition. Manipulation of brainstem nuclei with known inputs to the RVLM (nucleus tractus solitarius and caudal VLM) unmasked tonic glycinergic inhibition in the RVLM. Whole‐cell, patch clamp recordings demonstrate that both GABA and glycine inhibit RVLM neurons. Potentiation of neurotransmitter release from the active synaptic inputs in the RVLM produced saturation of GABAergic inhibition and emergence of glycinergic inhibition. Our data suggest that GABA controls threshold excitability, wherreas glycine increases the strength of inhibition under conditions of increased synaptic activity within the RVLM. Abstract The arterial baroreflex is a rapid negative‐feedback system that compensates changes in blood pressure by adjusting the output of presympathetic neurons in the rostral ventrolateral medulla (RVLM). GABAergic projections from the caudal VLM (CVLM) provide a primary inhibitory input to presympathetic RVLM neurons. Although glycine‐dependent regulation of RVLM neurons has been proposed, its role in determining RVLM excitability is ill‐defined. The present study aimed to determine the physiological role of glycinergic neurotransmission in baroreflex function, identify the mechanisms for glycine release, and evaluate co‐inhibition of RVLM neurons by GABA and glycine. Microinjection of the glycine receptor antagonist strychnine (4 mm, 100 nL) into the RVLM decreased the duration of baroreflex‐mediated inhibition of renal sympathetic nerve activity (control = 12 ± 1 min; RVLM‐strychnine = 5.1 ± 1 min), suggesting that RVLM glycine plays a critical role in regulating the time course of sympathoinhibition. Blockade of output from the nucleus tractus solitarius and/or disinhibition of the CVLM unmasked tonic glycinergic inhibition of the RVLM. To evaluate cellular mechanisms, RVLM neurons were retrogradely labelled (prior injection of pseudorabies virus PRV‐152) and whole‐cell, patch clamp recordings were obtained in brainstem slices. Under steady‐state conditions GABAergic inhibition of RVLM neurons predominated and glycine contributed less than 25% of the overall inhibition. By contrast, stimulation of synaptic inputs in the RVLM decreased GABAergic inhibition to 53%; and increased glycinergic inhibition to 47%. Thus, under conditions of increased synaptic activity in the RVLM, glycinergic inhibition is recruited to strengthen sympathoinhibition. - 'The Journal of Physiology, EarlyView. '