We previously found that the thiol amino acid L-cysteine microinjected into rat medullary autonomic areas produces changes in arterial blood pressure (AP) via ionotropic excitatory amino acid receptors (iEAAr), but its effect on vascular beds is still unknown. Rostral ventrolateral medulla (RVLM) pressor area includes adrenal and lumbar presympathetic neurons which activation could cause opposite muscle vascular responses: vasodilation versus vasoconstriction. However, there are few data on the vasodilator response in rats. Ionotropic EAAr activation alone with L-cysteine may be effective to differentiate RVLM sites for those opposite responses. To test it, muscle blood flow responses to L-cysteine were mapped in the ventrolateral medulla (VLM) of rats. In anesthetized rats with a cranial window above the VLM, hindquarter flow (HQF) and AP were monitored, providing hindquarter resistance (HQR) by dividing AP by HQF. L-Cysteine mapping in VLM including caudal depressor vasodilator area defined with L-glutamate showed HQR responses in parallel to AP responses, suggesting the importance of iEAAr in muscle vascular regulation. Microinjections of L-cysteine into RVLM succeeded to detect sites of slower muscle vasodilation and blockade of peripheral β-adrenoceptors abolished this response, indicating potential adrenaline secretion. Although there was no functional topography, the iEAAr activation alone with L-cysteine can differentiate sites of muscle vasodilation from vasoconstriction in rat RVLM. The neuromodulator candidate L-cysteine is a useful tool when chemical stimulation of iEAAr is required.