The present study aimed to characterize microcirculatory responses to anesthesia in brain tissue. With multi-photon excitation fluorescence microscopy, intra-cortical capillary dimension and red blood cell (RBC) flow were successfully visualized up to a depth of approximately 0.6 mm from the cortical surface in rats anesthetized with either isoflurane or alpha-chloralose. We observed that the diameter of the major cerebral artery was approximately 100 microm under isoflurane, but approximately 75 microm under alpha-chloralose. The capillary diameter was observed to be larger under alpha-chloralose than isoflurane: 5.1 +/- 1.2 microm vs. 4.8 +/- 1.1 microm, respectively. A significant difference in the mean RBC speed measured in single capillaries was observed: 0.4 +/- 0.4 mm/s under alpha-chloralose vs. 1.5 +/- 0.4 mm/s under isoflurane. In agreement with these observations, arterio-venous transit-time and laser-Doppler flowmetry consistently showed a significant reduction of the RBC and plasma blood speed under alpha-chloralose relative to isoflurane. These findings may indicate that local blood flow regulatory mechanisms exist at the capillary level for the balance of oxygen supply and demand induced by anesthesia in the brain tissue.