In this communication, we report the imaging of living glioma cells using fluorescence lifetime imaging (FLIM) technique. The growing interests in developing novel techniques for diagnosis and minimally invasive therapy of brain tumor have led to microscopic studies of subcellular structures and intracellular processes in glioma cells. Fluorescence microscopy has been used with a number of exogenous molecular probes specific for certain intracellular structures such as mitochondria, peripheral benzodiazepine receptor (PBR), and calcium concentration. When probes with overlapping emission spectra being used, separate samples are required to image each probe individually under conventional fluorescence microscopy. We have developed a wide-field FLIM microscope that uses fluorescence lifetime as an additional contrast for resolving multiple markers in the same essay. The FLIM microscope consists of a violet diode laser and a nitrogen-pumped dye laser to provide tunable sub-nanosecond excitation from UV to NIR. The detection system is based on a time-gated ICCD camera with minimum 80 ps gate width. The performance of the system was evaluated using fluorescence dyes with repotted lifetime values. Living rat glioma C6 cells were stained with JC-1 and Rhodamine 123. FLIM images were acquired and their lifetimes in living cells were found in good agreements with values measured in solutions by a time-domain fluorescence spectrometer. These results indicate that imaging of glioma cells using FLIM can resolve multiple spectrally-overlapping probes and provide quantitative functional information about the intracellular environment.