Dispersion of particulate nano-fillers has been assumed to be one of the most important determining factors of physical properties of the composites. In this paper, for the first time, a three dimensional (3D) observation of nano-structure of particulate silicas in natural rubber (NR) is reported by using a 3D transmission electron microscope (3D-TEM), which is a TEM combined with electron tomography to reconstruct 3D structural images. Here, in situ silica generated in NR matrix and a conventional silica (VN-3) mechanically mixed into NR were, without any staining of the samples, subjected to elucidation of their structural characteristics such as particle size, volume fraction and aspect ratio in NR matrix at nanometer scale. 3D-TEM/electron tomography enabled us to evaluate the density of in situ silica in NR matrix. The same samples were also subjected to atomic force microscopy (AFM) measurements to suggest a difference in the nano-filler/rubber interface area between the two samples. Since the dispersion of nano-filler in the amorphous matrix significantly controls the properties of nano-composites, 3D-TEM/electron tomography together with AFM is expected to play a most important role in revealing nanometer level 3D structure of soft materials. © 2005 Elsevier Ltd. All rights reserved.