MRI is a powerful tool for measuring cerebral blood flow (CBF) longitudinally. However, most animal studies require anesthesia, potentially interfering with normal physiology. Isoflurane anesthesia was used here to study CBF regulation during repetitive scanning in rats. MR perfusion images were acquired using FAIR (flow-sensitive alternating inversion recovery) arterial spin labeling, and absolute CBF was calculated. CBF changes in response to a hypoxic (12% O2) and hypercapnic (5% CO2) gas stimulus were monitored. Hypercapnia led to a robust increase in CBF compared with baseline (195.5+/-21.5 vs 123.6+/-17.9 ml/100 g/min), and hypoxia caused a smaller non-significant increase in mean CBF values (145.4+/-13.4 ml/100 g/min). Strikingly, when measurements were repeated 5 days later, CBF was dramatically reduced in hypoxia (93.2+/-8.1 ml/100 g/min) compared with the first imaging session. Without application of the hypoxic and hypercapnic gases during the first MRI, baseline CBF and CBF changes in response to hypoxia at the second MRI were similar to naive rats. Blood gas analyses revealed a slight reduction in arterial oxygenation during the second period of anesthesia compared with the first. These findings indicate that, in isoflurane-anesthetized rats, even a short hypoxic episode can have long-lasting effects on cerebrovascular regulation.