PURPOSE. The aim of this study was to investigate the autofluorescence (AF) of the RPE with two-photon microscopy (TPM) and fluorescence lifetime imaging (FLIM) under normal and oxidative stress conditions. METHODS. Porcine RPE-choroid explants were used for investigation. The RPE-choroid tissue was preserved in a perfusion organ culture system. Oxidative stress was induced by laser photocoagulation with frequency-doubled Nd:YAG laser (532 nm) and by exposure to different concentrations (0, 1, 10 mM) of ferrous sulfate (FeSO4) for 1 hour. At indicated time points after exposure, the tissue was examined with TPM and FLIM. Intracellular reactive oxygen species around the photocoagulation lesion were detected with chloromethyl-2'7'- dichlorofluorescein diacetate (CM-H2DCFDA). Melanosomes were isolated from RPE cells and their fluorescence properties were investigated under normal and oxidized conditions. RESULTS. Under normal conditions, AF in RPE cells with TPM is mostly originated from melanosomes, which has a very short fluorescence lifetime (FLT; mean = 117 ps). Under oxidative stress induced by laser irradiation and FeSO4 exposure, bright granular AF appears inside and around RPE cells, whose FLT is significantly longer (mean = 1388 ps) than the FLT of the melanosome-AF. Excitation and emission peaks are found at 710 to 750 nm and 450 to 500 nm, respectively. Oxidative stress increases the fluorescence intensity of the melanosomes but does not change their FLT. CONCLUSIONS. TPM reveals acute oxidative stress-induced bright AF granules inside and around RPE cells which can be clearly discriminated from melanosomes by FLIM. TPM combined with FLIM is a useful tool of live-cell analysis to investigate functional alterations of the RPE.