Several techniques are nowadays available to determine the size distribution of nanoparticulate matter. Among these techniques, atomic force microscopy (AFM) is especially valuable because it can provide three-dimensional information on the shape of individual nanoparticles. This paper describes a new method to determine the size distribution of a population of spherical nanoparticles deposited on a hard substrate. The method is based on the acquisition and analysis of topographical AFM images. The size of individual nanoparticles is obtained by fitting the topographical region associated with the nanoparticle with a sphere. Tests on model systems based on nanoparticle reference materials consisting of polystyrene (PS) latex suspensions show promising results. The measured mean particle size is larger than the reference value, but this is a predictable effect of the AFM tip shape. Tests on a bi-modal mixture of two PS latex reference materials show the impact of the quality of the dispersion of the nanoparticles on the results obtained with the new technique.JRC.D.2-Reference material