Sulfonated meso-tetraphenylporphyrins of different hydrophilicity were microspectrofluorimetrically examined in endothelial cells using total internal reflection (TIR) illumination or epi-illumination. Since the penetration depth of the evanescent field during TIR illumination is limited to a few hundred nanometers, photosensitizers were almost selectively examined in close vicinity to the plasma membrane. Pronounced fluorescence signals during TIR illumination were observed for the hydrophilic compounds meso-tetraphenylporphyrin tetrasulfonate (TPPS₄) and meso-tetraphenylporphyrin trisulfonate (TPPS₃), whereas the more lipophilic compounds meso-tetraphenylporphyrin disulfonate (TPPS_2a) and meso-tetraphenylporphyrin monosulfonate (TPPS₁) could only be detected under epi-illumination. Irradiation of TPPS₁ and TPPS_2a in the Soret band led to an increase in fluorescence intensity and formation of a photoproduct with an emission maximum around 610 nm, which was limited to intracellular compartments. In contrast, fluorescence spectra of TPPS₃ and TPPS₄ obtained by TIR and epi-illumination remained almost unchanged after irradiation in the Soret band. Extralysosomal location of TPPS₃ and TPPS₄ in close proximity to the plasma membrane was deduced from experiments with the lysosomal markers acridine orange (AO) or lysotracker yellow (LY), which were not detectable under TIR illumination. In conclusion, these results provide for the first time direct evidence for a plasma membrane-associated fraction of the hydrophilic compounds TPPS₃ and TPPS₄ in living cells.