Noninvasive and reliable quantification of rheological char-acteristics in the nucleus is extremely useful for fundamental research and practical applications in medicine and biology. This study exam-ines the use of fluorescence correlation spectroscopy (FCS) to nonin-vasively determine nucleoplasmic viscosity(ηnu), an important pa-rameter of nucleoplasmic rheology. Our FCS analyses show thatnu of lung adenocarcinomaASTC-a-1 and HeLa cells are 1.77±0.42 cP and 1.40±0.27 cP, respectively, about three to four times larger than the water viscosity at 37℃.ηnu was reduced by 31 to 36% upon hypotonic exposure and increased by 28 to 52% from 37 to 24℃. In addition, we found that ηnu of HeLa cells reached the lowest value in the S phase and that there was no significant differ-ence of ηnu between in the G1 and G2 phases. Last, nucleoplasmic viscosity was found to be larger than cytoplasmic viscosity in both HeLa and ASTC-a-1 cells. These results indicate that FCS can be used as a noninvasive tool to investigate the microenvironment of living cells. This is the first report on the measurement of ηnu in living cells synchronized in the G1, S, and G2 phases.