Multiphoton excitation fluorescent microscopy is a laser-based technology that allows subcellular resolution of native tissues in situ. We have recently applied this technology to the structural and photochemical imaging of cultured glioma cells and experimental gliomas ex vivo. We demonstrated that high microanatomical definition of the tumor, invasion zone, and normal adjacent brain can be obtained down to single-cell resolution in unprocessed tissue blocks. In this study, we used multiphoton excitation and four-dimensional microscopy to generate fluorescence lifetime maps of the murine brain anatomy, experimental glioma tissue, and biopsy specimens of human glial tumors. In murine brain, cellular and noncellular elements of the normal anatomy were identified. Distinct excitation profiles and lifetimes of endogenous fluorophores were identified for specific brain regions. Intracranial grafts of human glioma cell lines in mouse brain were used to study the excitation profiles and fluorescence lifetimes of tumor cells and adjacent host brain. These studies demonstrated that normal brain and tumor could be distinguished on the basis of fluorescence intensity and fluorescence lifetime profiles. Human brain specimens and brain tumor biopsies were also analyzed by multiphoton microscopy, which demonstrated distinct excitation and lifetime profiles in glioma specimens and tumor-adjacent brain. This study demonstrates that multiphoton excitation of autofluorescence can distinguish tumor tissue and normal brain based on the intensity and lifetime of fluorescence. Further technical developments in this technology may provide a means for in situ tissue analysis, which might be used to detect residual tumor at the resection edge.