Size is the most studied parameter in the field of nanoparticle characterization but few studies have been performed on biodegradable particles with well-defined sizes. The aim of this work was to prepare fluorescent biodegradable polymeric particles having well-defined sizes and well-characterized surface properties. Poly(D,L-lactide-co-glycolide acid) particles were prepared by the emulsion evaporation process. Filtration and centrifugation were used to produce particle fractions in the narrow size range from polydispersed batches, and the efficiency of separation was compared. Selective centrifugation allowed for the preparation of five classes of particles having narrow size distribution (0.1, 0.3, 0.6, 1 and 2 microm). Particles were characterized in terms of size distribution, surface morphology, charge, residual surfactant and hydrophilicity. The results showed similar surface properties for all the batches. 3,3'-Dioctadecyloxacarbo-cyanine perchlorate has been successfully incorporated as a fluorescent dye and its ability to remain associated with the particles during cell culture experiments has been proven. Such particles may be used as an adequate tool for studying cellular uptake.