We have developed a series of intensely-stained fluorescent microspheres, FluoSpheres®, for research in regional blood flow determination. FluoSpheres are uniform polystyrene microspheres with nominal diameters of 10 µm and 15 µm. These microspheres have a relative density of 1.05 g/mL, which is close to that of red blood cells (1.10 g/mL). We have prepared FluoSpheres with seven different excitation/emission wavelength combinations, each spectrally distinct from the others. This allows effects of multiple physiological variables to be studied in the course of a single experiment. All of the microparticles are uniformly stained, using a proprietary process that results in incorporation of the dye in the microsphere interior. Compared to surface staining, this method has two important advantages. Firstly, more dyes can be incorporated per bead (since volume>surface area), resulting in greater fluorescence per particle. Secondly, the dye is shielded from contact with caustic reagents used in tissue extraction processes and also from reactive oxygen species that cause photobleaching. Our quality control data indicate that FluoSpheres microparticles have a shelf life of at least 18 months and do not show any spectral or fluorescence intensity changes after several temperature change cycles. The long term stability of FluoSpheres is in marked contrast to the limited shelf life of radioactively-labeled microspheres.