In order to localize groups of neurons commanding the defense reaction, microinjections of L-glutamate (GLU, 5 nmol in 0.2 microliter, during 20 s) were made inside the dorsal midbrain of unanesthetized rats provided with an intra-arterial cannula for blood pressure (BP) recording. In 9 rats, GLU microinjection induced freezing behavior or flight, accompanied by a 30.1 +/- 4.8 mmHg increase in BP. The latency of the GLU effect was 16 +/- 2 s and its duration 89 +/- 8 s, as measured from the BP recordings. In 9 other rats, GLU microinjection did not evoke defensive behavior and the BP increase was 6.0 +/- 0.9 mmHg, significantly lower (P less than 0.001) than in the former group. Control injections of artificial cerebrospinal fluid (CSF) did not cause behavioral changes and increased BP by less than 3 mmHg. Histology revealed that in all rats in which GLU induced a defense reaction the injection sites were inside the dorsal periaqueductal gray (PAG). Sites in which GLU injection was ineffective were localized in the midbrain tegmentum, outside the borders of the PAG (8 rats), or inside the ventral PAG (1 rat). Since GLU stimulates neuron cell bodies and their dendritic processes but not passing axons, these results strongly suggest that the dorsal PAG of the rat contains a group of neurons that controls behavioral and autonomic manifestations of the defense reaction.