Urocortin 3 (Ucn 3) is a new member of the corticotrophin-releasing factor (CRF) peptide family and is considered to be a specific and endogenous ligand for the CRF type 2 receptors (CRF2Rs). The presence of CRF₂Rs has been reported in the nucleus tractus solitarius (NTS) of the rat. It was hypothesized that activation of CRF₂Rs in the mNTS may play a role in cardiovascular regulation. This hypothesis was tested in urethane-anesthetized, artificially ventilated, adult male Wistar rats. Microinjections (100 nl) of Ucn 3 (0.03, 0.06, 0.12 and 0.25 mM) into the medial nucleus tractus solitarius (mNTS) of anesthetized rats elicited decreases in mean arterial pressure (MAP; 5.0 ± 1.0, 21.6 ± 2.6, 20.0 ± 2.8 and 12.7 ± 3.4 mmHg, respectively) and heart rate (HR; 7.8 ± 2.6, 46.2 ± 9.3, 34.5 ± 8.4 and 16.6 ± 4.9 bpm, respectively). Microinjections of artificial cerebrospinal fluid (100 nl) into the mNTS did not elicit cardiovascular responses. Maximum decreases in MAP and HR were elicited by 0.06 mM concentration of Ucn 3. Cardiovascular responses to Ucn 3 were similar in unanesthetized mid-collicular decerebrate rats. Bilateral vagotomy completely abolished Ucn 3-induced bradycardia. The decreases in MAP and HR elicited by Ucn 3 (0.06 mM) were completely blocked by astressin (1 mM; non-selective CRFR antagonist) and K41498 (5 mM; selective CRF₂R antagonist). Microinjections of Ucn 3 (0.06 mM) into the mNTS decreased efferent greater splanchnic nerve activity. After the blockade of CRF₂Rs in the mNTS, Ucn 3-induced decrease in efferent sympathetic nerve discharge was abolished. These results indicate that Ucn 3 microinjections into the mNTS exert excitatory effects on mNTS neurons via CRF₂Rs, leading to depressor and bradycardic responses.