As endothelial cells are subject to flow shear stress, it is important to determine the detailed velocity distribution in microvessels in the study of mechanical interactions between blood and endothelium. Recently, particle image velocimetry (PIV) has been proposed as a quantitative method of measuring velocity fields instantaneously in experimental fluid mechanics. The authors have developed a highly accurate PIV technique with improved dynamic range. spatial resolution and measurement accuracy. In this paper, the proposed method was applied to images of the arteriole in the rat mesentery using an intravital microscope and high-speed digital video system. Taking the mesentery motion into account, the PIV technique was improved to measure red blood cell (RBC) velocity. Velocity distributions with spatial resolutions of 0.8 x 0.8 microm were obtained even near the wall in the centre plane of the arteriole. The arteriole velocity profile was blunt in the centre region of the vessel cross-section and sharp in the near-wall region. Typical flow features for non-Newtonian fluid were shown. Time-averaged velocity profiles in six cross sections with different diameters were compared.