Hydrogen sulfide (H(2)S) is now recognized as an important signaling molecule and has been shown to have vasodilator and cardio-protectant effects. More recently it has been suggested that H(2)S may also act within the brain to reduce blood pressure (BP). In the present study we have demonstrated the presence of the H(2)S-producing enzyme, cystathionine-β-synthase (CBS) in the rostral ventrolateral medulla (RVLM), and the hypothalamic paraventricular nucleus (PVN), brain regions with key cardiovascular regulatory functions. The cardiovascular role of H(2)S was investigated by determining the BP, heart rate (HR), and lumbar sympathetic nerve activity (LSNA) responses elicited by a H(2)S donor sodium hydrogen sulfide (NaHS) or inhibitors of CBS, microinjected into the RVLM and PVN. In anesthetized Wistar Kyoto rats bilateral microinjections of NaHS (0.2-2000 pmol/side) into the RVLM did not significantly affect BP, HR, or LSNA, compared to vehicle. Similarly, when the CBS inhibitors, amino-oxyacetate (AOA; 0.1-1.0 nmol/side) or hydroxylamine (HA; 0.2-2.0 nmol/side), were administered into the RVLM, there were no significant effects on the cardiovascular variables compared to vehicle. Microinjections into the PVN of NaHS, HA, and AOA had no consistent significant effects on BP, HR, or LSNA compared to vehicle. We also investigated the cardiovascular responses to NaHS microinjected into the RVLM and PVN in spontaneously hypertensive rats. Again, there were no significant effects on BP, HR, and LSNA. Together, these results suggest that H(2)S in the RVLM and PVN does not have a major role in cardiovascular regulation.