The three-dimensional transmission electron microscope (3D-TEM) images of nano-filler-filled rubber vulcanizates were very low contrast. As one of the curing reagents, zinc oxide, and its derivatives formed during cross-linking reactions were assumed to be responsible for it. A removal method by extraction of the rubber-soluble zinc compounds, and so on by a solvent mixture was developed (NARC-AK method), and it was found to give clear 3D-TEM images. These images afforded quantitative analyses of nano-filler aggregates in the rubbery matrix. The 3D-TEM analysis on isoprene rubber (IR) loaded with HAF, FEF or FT was carried out before and after their vulcanization. The unique ratio (S_CB/V_CB) of the total surface without the six side areas of the 3D-TEM cuboidal image to the total volume of CB aggregate, was obtained from a 3D-TEM image, and activation energy (ΔE(T₂)) of spin-spin relaxation was measured on the immobilized rubber layer around CB from pulsed NMR measurements. A linear relationship between ΔE(T₂) and S_CB/V_CBwas observed. Also, a linear relationship was observed between the volume ratio of IR gel and S_CB/V_CB. The slopes of this approximate straight line gave an approximately 3.9 nm within experimental errors. This value is estimated to be equivalent to the thickness of bound rubber encircling the CB aggregates. The intercept value, approximately 4 wt%, may be interpreted to be suggesting that the IR gel fraction has changed little before and after the vulcanization.