Various microscopy techniques were employed to measure subcellular porphyrin distribution in ALA-incubated tumor cells and tumor spheroids. In addition, photodynamically-induced intracellular changes in morphology and fluorescence were detected. Studies were carried out by confocal laser scanning microscopy, polarization microscopy, video-intensified microscopy, video-enhanced contrast microscopy, time-resolved and time-gated spectroscopy, and spectrally-resolved fluorescence imaging using a tunable liquid crystal filter. It was found that ALA-incubation results in the biosynthesis of protoporphyrin IX (PP IX) in the mitochondria. Long ALA incubation times (>6 h) results in PP IX release to the cytoplasm. PP IX fluorescence has a maximum at 635 nm and a fluorescence decay time of 16 ns. No evidence for the existence of short-lived dimers, aggregates of PP IX, or other porphyrins was found. Cell irradiation resulted in cytotoxic reactions which, in turn, led to mitochondrial swelling followed by destruction of the cell membrane. In addition, PP IX photodestruction and the formation of short-lived chlorin-type photoproducts were observed.