Anesthetics, widely used in magnetic resonance imaging (MRI) studies to avoid movement artifacts, could have profound effects on cerebral blood flow (CBF) and cerebrovascular coupling relative to the awake condition. Quantitative CBF and tissue oxygenation (blood oxygen level-dependent [BOLD]) were measured, using the continuous arterial-spin-labeling technique with echo-planar-imaging acquisition, in awake and anesthetized (2% isoflurane) rats under basal and hypercapnic conditions. All basal blood gases were within physiologic ranges. Blood pressure, respiration, and heart rates were within physiologic ranges in the awake condition but were depressed under anesthesia (P < 0.05). Regional CBF was heterogeneous with whole-brain CBF values of 0.86 +/- 0.25 and 1.27 +/- 0.29 mL. g-1. min-1 under awake and anesthetized conditions, respectively. Surprisingly, CBF was markedly higher (20% to 70% across different brain conditions) under isoflurane-anesthetized condition compared with the awake state (P < 0.01). Hypercapnia decreased pH, and increased Pco(2) and Po(2). During 5% CO(2) challenge, under awake and anesthetized conditions, respectively, CBF increased 51 +/- 11% and 25 +/- 4%, and BOLD increased 7.3 +/- 0.7% and 5.4 +/- 0.4%. During 10% CO(2) challenge, CBF increased 158 +/- 28% and 47 +/- 11%, and BOLD increased 12.5 +/- 0.9% and 7.2 +/- 0.5%. Since CBF and BOLD responses were substantially higher under awake condition whereas blood gases were not statistically different, it was concluded that cerebrovascular reactivity was suppressed by anesthetics. This study also shows that perfusion and perfusion-based functional MRI can be performed in awake animals.