The neurotransmitter candidate L-proline elicits changes in the cardiovascular system via actions in the brainstem. However, its action have not yet been determined in the ventrolateral medulla (VLM), a brain region critical in mediating vasomotor sympathetic nervous system responses. Microinjections of L-glutamate produce depressor responses in the caudal (C) VLM, but pressor responses in the rostral (R) VLM and the caudal pressor area (CPA) in the far caudal CVLM. The present study tested whether microinjections of l-proline in the VLM produce a pattern of hemodynamic responses distinct from that of l- glutamate. Urethane-anesthetized rats received arterial catheters and were implanted with flow probes around the abdominal aorta (supplies hindquarters). The surface of each rat's VLM was then exposed. L-Proline induced dose- dependent depressor responses in the CVLM (0.003–1.0 M, 34 nl), but did not induce hemodynamic responses in sites of the RVLM (0.01–1.0 M, 34 nl) that responded to L-glutamate (0.01 M, 34 nl). L-Proline injections (0.1 M, 34 nl) induced rapid and consistent depressor responses correlated with coincident decreases in hindquarter resistance (arterial blood pressure/flow) in the CVLM and CPA, but only inconsistent responses in a few sites in the RVLM. In summary, L-proline induced a distinct pattern of depressor responses preferentially in caudal regions of the VLM, and these depressor effects were associated with decreases in hindquarter resistance. These findings indicate that l-proline may have unique roles including cardiovascular regulation independently from L-glutamate, especially in caudal region of the VLM, via a mechanism that involves altering hindquarter resistance.