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Cells are frequently interconnected by tunneling nanotubules, recently discovered cylindrical structures which facilitate material exchange. We employed here single molecule fluorescence microscopy to study the diffusion of the glycosylphosphatidylinositol-protein CD59 in the plasma membrane of tunneling nanotubules in living cells at subwavelength resolution. Our study provides the nanotubule radius in vivo, yielding a surprisingly narrow size distribution (7nm standard deviation) around a mean value of 65nm. Moreover, by separating longitudinal and transverse mobilities, we find isotropic diffusion behavior.