The widespread and abundant distribution of P2Y receptors in the mammalian brain suggests important functions for these receptors in the CNS. To study a possible involvement of the P2Y receptors in the regulation of fear and anxiety, the influences of the P2Y 1, 11, 12 receptor-specific agonist adenosine 5′-O-(2-thiodiphosphate)(ADPβS), the P2X 1, 3 receptor agonist α, β-methylene ATP (α, βmeATP), the unspecific P2 receptor antagonist pyridoxalphosphate-6-azopheny l-2′, 4′-disulfonic acid (PPADS), and the specific P2Y 1 receptor antagonist N 6-methyl-2′-deoxyadenosine-3′, 5′-bisphosphate (MRS 2179) on the elevated plus-maze behavior of the rat were investigated. All tested compounds were given intracerebroventricularly (0.5 μl). ADPβS (50 and 500 fmol) produced an anxiolytic-like behavioral profile reflected by an increase of the open arm exploration. The anxiolytic-like effects were antagonized by pretreatment with PPADS (5 pmol) or MRS 2179 (5 pmol). Both compounds caused anxiogenic-like effects when given alone. Furthermore, the anxiolytic-like effects of ADPβS could be antagonized by pretreatment with the nitric oxide synthase (NOS) inhibitor N w-nitro-L-arginine methyl ester (L-NAME). In addition, the anxiogenic-like effects of PPADS were reversed by the pretreatment with L-arginine (500 pmol), which is the natural substrate for NOS, but not by D-arginine (500 pmol), which is not. Immunofluorescence staining revealed the presence of P2Y 1 receptors on neurons in different brain regions such as hypothalamus, amygdala, hippocampus and the periaqueductal gray. Furthermore, the colocalization of P2Y 1 receptors and neuronal NOS (nNOS) on some neurons in these regions could be demonstrated. The highest density of P2Y 1-and nNOS-immunoreactivity was detected in the dorsomedial hypothalamic nucleus. Taken together, the present results suggest that P2Y 1 receptors are involved in the modulation of anxiety in the rat. The anxiolytic-like effects after stimulation of P2Y 1 receptors seem to be in close connection with the related nitric oxide production.