Optical spectroscopy for brain tumor demarcation was investigated in this study. Fluorescence and diffuse reflectance spectra were measured from normal and tumorous human brain tissues in vitro. A fluorescence peak was consistently observed around 460 nm (±10 nm) emission from both normal and tumorous brain tissues using 337 nm excitation. Intensity of this fluorescence peak (F460) from normal brain tissues was greater than that from primary brain tumorous tissues. In addition, diffuse reflectance (Rd) between 650 and 800 nm from white matter was significantly stronger than that from primary and secondary brain tumors. A good separation between gray matter and brain tumors was found using the ratio of F460 and Rd at 460 nm (Rd460). Two empirical discrimination algorithms based on F460,Rd625, and F460/Rd460 were developed. These algorithms yielded an average sensitivity and specificity of 96% and 93%, respectively. © 2000 Society of Photo-Optical Instrumentation Engineers.