The dorsolateral periaqueductal gray matter (DLPAG) is a key structure in behavioral and autonomic expression of defensive reactions. Inhibitors of nitric oxide (NO) synthase microinjected into the DLPAG are anxiolytic. The purpose of the present study was to evaluate the behavioral effects produced by administration of two NO donors, SIN-1 or DEA/NO, into the DLPAG. We also employed the detection of Fos-like immunoreactivity (FLI) to reveal brain areas activated by SIN-1 administration. Male Wistar rats (n = 7-11/group) received intra-DLPAG injection of SIN-1 (150 or 300 nmol), DEA/NO (150, 300, or 600 nmol), or saline (0.5 microl), and their behavior was observed in an open arena during 15 min. For the FLI assay, the animals (n = 3-5/group) were sacrificed 2.25 h after the drug. In a second experiment, rats (n = 5-7/group) received a first intra-DLPAG injection of saline or methylene blue (MB, 30 nmol), an NO antagonist, followed by saline or SIN-1 (300 nmol). SIN-1 (300 nmol) induced a flight response characterized by coordinated running and oriented jumps with escape attempts. Similar but short-lasting changes were seen after the administration of DEA/NO. FLI was dose-dependently induced by SIN-1 in several regions related to defensive reactions, including the periaqueductal gray, hypothalamic nuclei, medial amygdala, and cingulate cortex (analysis of variance, p < 0.05). A greater number of neurons showing FLI was found ipsilateral to the microinjection site. The drug effect was greater at this side in the bed nucleus of the stria terminalis, paraventricular and lateral hypothalamus, cingulate cortex, septohipoccampal nucleus, and horizontal limb of the diagonal band. The increase in SIN-1-induced FLI was attenuated by MB pretreatment in most regions. These results suggest that NO may participate in the modulation of defensive responses in the DLPAG.