Subcellular microenvironments within a living cell are expected to intimately correlate with cellular events including cell division, phagocytosis, locomotion, and intracellular transport. Thus, understanding diffusion processes of macromolecules in subcellular compartments is important to elucidate functions of these compartments. Some biologically active molecules are reported to move by Brownian motion at low mobility due to association and dissociation of cellular components. Measurement of diffusion of inert macromolecules including green fluorescent protein (GFP) is also important to evaluate anomalous diffusion of biologically active macromolecules. Here, we analyzed mobility of GFP in the nucleoplasm, nucleolus, and cytoplasm using fluorescence correlation spectroscopy (FCS). GFP molecules were transiently expressed in HeLa cells, and distributed widely throughout the nucleus. FCS analysis indicated that GFP molecules move by Brownian motion with two different diffusion coefficients. About 97% of GFP in the nucleoplasm and 87% of GFP in the nucleolus moved with diffusion coefficients of 68 μm²⁄s and 45 μm²⁄s at 37°C, respectively. These diffusion coefficients were 2 to 3-fold smaller than that in solution (126 μm²⁄s). The remaining GFP molecules moved slower at 3.8 μm²⁄s in the nucleoplasm, and 4.5 μm²⁄s in the nucleolus. Diffusion coefficients decreased to 28 μm²⁄s in the nucleoplasm, and to 12 μm²⁄s in the nucleolus at 23°C. Amount of slow components decreased to 3% in the nucleolus, and was undetectable in the nucleoplasm at 23°C. These results indicated that diffusion of protein molecules was different in different nuclear microstructures of a living cell, and was temperature-dependent.