Fourier transform multipixel spectroscopy was applied to subcellular localization of endogenous protoporphyrin (endo-PP) in single living B16 melanoma cells during photosensitization. Continuous fluorescence spectra for each pixel were recorded using a Sagnac interferometer coupled to a charge-coupled device camera. Multiple frames of data were acquired for each pixel composing the image, then they were stored as interferometric data and resolved as spectra for every pixel (10(3)-4 x 10(3) point pixels in a single cell). The net result was the intensity I (x, y, gamma), for each pixel of the image (x,y), at any wave-length (gamma). The present study demonstrates the application of Fourier transformed multipixel spectroscopy for spectral imaging of melanoma cells incubated with 5-aminolevulinic acid (ALA). The fluorescence image of ALA-treated cells revealed endo-PP all over the cytosol with a vesicular distribution, which represent mitochondria and endoplasmic reticulum compartments. Two main spectral fluorescence peaks were demonstrated at 630 and 670 nm, of monomeric and aggregated protoporphyrin, with intensities that differed from one subcellular site to another. Photoirradiation of the cells induced point-specific subcellular fluorescence spectrum changes and demonstrated photoproduct formation. Spectral-image reconstruction revealed the subcellular distribution of porphyrin species in single photosensitized cells. Multipixel spectroscopy of exogenous protoporphyrin revealed an endosomal-lysosomal compartment in aggregated states, whereas monomeric porphyrin species were localized mainly on the outer membrane. Photo-products could be visualized at sites of formation in subcellular compartments.