IMMUNOHISTOCHEMICAL studies have shown nitric oxide synthase (NOS)-positive neurons in the dorsolateral sector of periaqueductal gray (PAG), a neural site known to be critical for the expression of defensive responses. In the present study, we first characterized the dorsal PAG (dPAG) NOS, and then examined NOS activity and cyclic GMP (cGMP) accumulation in the dPAG of rats exposed to a predator (cat) for 15 min. NOS activity evaluated by enzymatic conversion of [3H]arginine to [3H]citrulline in dPAG of exposed rats increased 14.6% and the cGMP radioimmunoassay showed an increase of 30.6% in relation to non-exposed rats. These results suggest an involvement of the NO/cGMP pathway in the dPAG during defensive responses.