We measured the changes in pial arteriolar diameter and CSF concentrations of adenosine, inosine, and hypoxanthine during hypoxia in the absence and presence of topically applied dipyridamole (10(-6) M) and erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA; 10(-5) M). Closed cranial windows were implanted in halothane-anesthetized adult male Sprague-Dawley rats for the observation of the pial circulation and collection of CSF. The mean resting arteriolar diameter in mock CSF was 31.2 +/- 5.9 microns. Topically applied dipyridamole and EHNA, in combination, caused a slight but significant (p < 0.05) increase in resting arteriolar diameter (33.8 +/- 4.3 microns). With mock CSF, moderate hypoxia caused a 22.1 +/- 9.7% increase in pial vessel diameter. Topically applied dipyridamole and EHNA significantly (p < 0.01) potentiated pial arteriolar vasodilation in response to hypoxia. Moreover, the potentiating effects of dipyridamole and EHNA during hypoxia were completely abolished by theophylline (0.20 mumol/g, i.p.; p < 0.05), an adenosine receptor antagonist. Resting concentrations of adenosine, inosine, and hypoxanthine in the subwindow CSF were 0.18 +/- 0.09, 0.35 +/- 0.21, and 0.62 +/- 0.12 microM, respectively. In the absence of dipyridamole and EHNA, these levels were not affected by sustained moderate hypoxia (PaO2 = 36 +/- 6 mm Hg). However, in the presence of dipyridamole and EHNA, the concentration of adenosine in the CSF during hypoxia was significantly (p < 0.05) increased. Our data indicate that dipyridamole and EHNA potentiate hypoxic vasodilation of pial arterioles while simultaneously increasing extracellular adenosine levels, thus supporting the hypothesis that adenosine is involved in the regulation of cerebral blood flow.