This paper describes a complete sampling, fractionation and characterization scheme for submicrometer particles isolated from the Rhine River. Decreasing particle size fractions were obtained by means of gravitational sedimentation, cascade centrifugation/ultracentrifugation and cascade filtration. These size fractions were analyzed by photon correlation spectroscopy (PCS), micro-electrophoresis (ME), transmission electron microscopy (TEM), light scattering (LS), inductively coupled plasma-atomic emission spectrometry (ICP-AES) and total organic carbon (TOC), which gave complementary results concerning the nature of the particles. The data indicated that submicron particles contribute only a small proportion of the total particle mass and volume, but an important proportion of the total particle number. Moreover, their specific surface area may be quite large. Associations of submicrometer particles with organic macromolecules and fibrils, which may have maintained such particles in suspension, were observed.