L-Glutamate has been considered to be a neurotransmitter in the nucleus tractus solitarius (NTS) of the afferent baroreflex pathway, though this has not yet been decisively shown. A bolus injection of a neurotransmitter candidate amino acid L-proline into the cisterna magna and that of L-glutamate shows the same pressor action in the freely moving rat, but the actual nuclei responding L-proline remain undetermined. Besides L-glutamate, L-proline might be another candidate amino acid in the NTS. The present study was therefore performed to characterize the circulatory action of L-proline injected into the NTS where responses to glutamate in the anesthetized rat had already been shown. The NTS was first determined as a site on the dorsal surface of the medulla where a microinjection of L-glutamate decreased arterial pressure and heart rate. Microinjected L-proline (1.65 to 13.2 nmol, 33 nl) into the NTS decreased arterial pressure and heart rate in a dose-dependent manner. The injection of a mixed solution (66 nl) of kynurenate, an ionotropic excitatory amino acid receptors antagonist (1.32 nmol), and L-proline (6.6 nmol) into the NTS abolished the depressor and bradycardic actions with L-proline alone (6.6 nmol, 66 nl). However, a mixture of an increased concentration of kynurenate (6.6 nmol) with glutamate augmented the actions seen with glutamate alone (0.66 nmol, 66 nl). D-Proline (13.2 nmol, 66 nl), the optic isomer of L-proline, produced no change in arterial pressure or heart rate, suggesting that the actions of L-proline in the NTS were optically specific. The results indicate that L-proline but not D-proline induces its depressor and bradycardic actions through ionotropic excitatory amino acid receptors in the NTS of the anesthetized rat. L-Proline may become a candidate transmitter of baroreceptor information in the NTS.