Abnormalities in adenosinergic regulation of peripheral vascular function have been implicated in the development of hypertension in spontaneously hypertensive rats (SHR) (Illes et al., 1989). Here, we examined the role of adenosine receptors in regulating cerebrovascular reactivity in pre-hypertensive SHR, an animal model with high cerebrovascular resistance (Cates et al., 2011) and brainstem hypoperfusion (Marina et al., 2015). Changes in the diameter of pial arterioles (20-100 µm) in response to adenosine agonists were assessed after pre-constriction with the thromboxane agonist U46619 in 400-µm-thick horizontal slices from the cortex and ventral brainstem of 4-6 week-old Wistar and SHR. Measurements were made using a Leica confocal (SP) microscope. Values are means ± S.E.M., compared by ANOVA. U46619 (200 nM) induced a similar vasoconstriction in arterioles from Wistar and SHR. In brainstem slices, adenosine (10 µM) reversed the U46619-induced vasoconstriction in Wistar but not SHR (+41±5% vs. +10±5%, n=6/8, respectively, p<0.05). In contrast, in cortical slices, the vasodilator effect of adenosine was similar between Wistar and SHR (+45±3% and +47±2%, n=11/13, respectively). The selective A2A receptor agonist (CGS-21680, 1 µM) dilated pre-constricted brainstem arterioles from Wistar but not SHR (+31±9% vs. -9±7%, n=6/5, respectively, p<0.01), while its vasodilator effect in the cortex was similar between strains (+38±8% and +33±12%, n=8/8, respectively). Likewise, the vasodilator effect of the A1/A2 agonist 5′-N-ethylcarboxamidoadenosine (NECA, 1 µM) tended to be higher in brainstem slices from Wistar than SHR (+27±5% vs. +8±4%, n=7/7, respectively), while its efficiency in cortical slices was very similar between strains (Wistar: +54±11%; SHR: +55±10%, n=8/7, respectively). Pre-treatment with the selective A2A receptor antagonist ZM-241385 (0.1 µM) fully blocked the vasodilator effects of adenosine, CGS-21680 and NECA. In contrast to A2 receptor-related compounds, the selective A1 receptor agonist N6-Cyclopentyladenosine (1 µM) produced moderate vasoconstriction of a similar magnitude in the brainstem of Wistar and SHR (-17±7% and -24±9%, n=5/6, respectively), but no change in the cortex (+3±17% vs +1±8%, n=6/6, respectively). These results suggest that the vasodilator efficiency of adenosine A2A receptors is attenuated in the brainstem but not cortical arterioles of pre-hypertensive SHR. This brainstem-specific reduction in A2A receptor sensitivity could contribute to inadequacies of functional hyperaemia and brainstem hypoperfusion in the SHR, which may trigger sympathetic activation and hypertension.