Methylene blue (MB) is a safe, low cost, and common blue dye remaining a popular choice for sentinel lymph node (SLN) mapping. However, the blue-dyed SLN in deep animal tissue could be seen only after a surgical management. Here the fluorescence properties of MB were investigated using a fluorescence spectrometer and a wavelength-resolved fluorescence spectral in vivo imaging system, and MB has been demonstrated for near-infrared (NIR) fluorescence mapping of SLN. When MB was injected intradermally into the second row breast of a rabbit, the lymphatic flow and axillary SLN could be observed directly through the NIR fluorescence emitted from the MB trapped in the deep tissue, which would eliminate the need for surgical management, and this fluorescence without any radioactivity was retained in the SLN for hours. The node also could be identified synchronously by the blue color and fluorescence of the MB after a surgical management. Using MB NIR fluorescence for SLN mapping may have great advantages over the traditional method. (c) 2008, The Society for Biotechnology, Japan. All rights reserved.