Particle tracking velocimetry provides a Lagrangian description of flow properties in the microcirculation. To determine the utility of fluorescent nanoparticles to provide Lagrangian coordinates, we tracked these particles both in vitro and in vivo. The particles had a neutral charge and fluorescence intensity greater than 1,000 times the PKH26-labeled red blood cells. At image acquisition rates of 60 frames per second, particles were tracked at velocities up to 4,000 microm/s. Morphometric changes reflecting streaking artifact were significant at velocities of 4,000 microm/s (P < 0.05), but not at lower velocities (P > 0.05). Intravital microscopy monitoring after intravenous injection of the particles demonstrated a circulation half-life that was inversely related to particle size: 500 nm nanoparticles demonstrated a smaller change in plasma concentration than larger particles. Regardless of the size of the particles, more than 50% of the recovered fluorescence was located in the liver. These results suggest that fluorescent nanoparticles provide a convenient and practical Lagrangian description of flow velocity in the microcirculation.