Our previous study showed the microinjection of drugs that influence the nitric oxide (NO)-mediated neurotransmission in the hippocampus impacts upon the anxiolytic-like effect of ethanol. In this study, we examined whether NO-dependent pathways of the dorsolateral periaqueductal gray (dlPAG) participate in the anxiolytic effect of ethanol in rats submitted to the elevated plus-maze test. We evaluated the impact on ethanol effects of the nitric oxide synthase (NOS) inhibitor 7-nitroindazole, the soluble guanylate cyclase inhibitor 1H-(1,2,4)-oxodiazolo (4,3-a) quinoxalin-1-one (ODQ), the cyclic guanylate monophosphate (cGMP) analogue 8-bromo-cGMP and the NO donor sodium nitroprusside. The results showed that ODQ and 7-nitroindazole increased the percentage of open arm entries and of time spent on open arms in the elevated plus maze in rats injected with ethanol at 1.0g/kg, a dose that did not produce anxiolysis per se. Conversely, 8-bromo-cGMP and sodium nitroprusside blocked the increased exploration of open arms exhibited by rats treated with a higher dose of ethanol (1.2g/kg). Taken together, the results suggest that the inhibition of NO-dependent pathways of the dlPAG enhances the anxiolytic effect of ethanol, whereas the activation of these pathways results in an opposite effect.