The role of excitatory amino acid (EAA) receptors in the rostral ventrolateral medulla (RVLM) in maintaining resting sympathetic vasomotor tone remains unclear. It has been proposed that EAA receptors in the RVLM mediate excitatory inputs both to presympathetic neurons and to interneurons in the caudal ventrolateral medulla (CVLM), which then provide a counterbalancing inhibition of RVLM presympathetic neurons. In this study, we tested this hypothesis by determining the effect of blockade of EAA receptors in the RVLM on mean arterial pressure (MAP), heart rate (HR), and renal sympathetic nerve activity (RSNA), after inhibition of CVLM neurons. In anesthetized rats, bilateral injections of muscimol in the CVLM increased MAP, HR, and RSNA. Subsequent bilateral injections of kynurenic acid (Kyn, 2.7 nmol) in the RVLM caused a modest reduction of approximately 20 mmHg in the MAP but had no effect, when compared with the effect of vehicle injection alone, on HR or RSNA. By approximately 50 min after the injections of Kyn or vehicle in the RVLM, the MAP had stabilized at a level close to its original baseline level, but the HR and RSNA stabilized at levels above baseline. The results indicate that removal of tonic EAA drive to RVLM neurons has little effect on the tonic activity of RVLM presympathetic neurons, even when inputs from the CVLM are blocked. Thus the tonic activity of RVLM presympathetic neurons under these conditions is dependent on excitatory synaptic inputs mediated by non-EAA receptors and/or the autoactivity of these neurons.