The mean centerline red blood cell (RBC) velocity of the rat pial artery was measured using an image-intensified high-speed (1000 frames/s) video camera system and RBCs labeled with fluorescein isothiocyanate (FITC). Some investigations measuring RBC velocity have been made in most organs, but the RBC velocity of the pial artery has not yet been measured with this system using FITC labeled RBC. After recording the emission of the FITC labeled RBC through a closed cranial window using this system, the authors analyzed the videotape. The movement of each individual RBC for several milliseconds over a distance of 50 microm could be pursued. The mean centerline RBC velocity in normal rats varied between 1.0 and 9.0 mm/s (most of the measurements we taken in vessels ranging between 20 and 80 microm in diameter). As the diameter of the pial artery becomes smaller, the blood flow rate (pi x (diameter/2)2 x (mean centerline velocity/1.6)) tends to become smaller. During CO2 inhalation, the pial artery diameter, mean centerline RBC velocity, and blood flow rate increased with statistical significance. Mean centerline RBC velocities in the cerebral microcirculation could not be measured directly with accuracy using the older methods (30 frames/s). However, this method is useful for investigation of the cerebral microcirculation and is considered to be applicable for studying the behavior of leukocytes or platelets, which will be examined in a subsequent study.