Photodynamic therapy (PDT) employing photosensiter N-aspartyl chlorin e6 (NPe6) can induce lysosome disruption and initiate the intrinsic apoptotic pathway. Bid, a member of the Bcl-2 family of proteins, is an essential regulator of apoptosis. Bid is normally found in the cytosol of healthy cells in an inactive form, activation of Bid depends on the cleavage of intact Bid into its truncated form of tBid. The resulting tBid then translocates into mitochondria and leads to disruption of organelles and release of apoptogenic molecules such as cytochrome c in response to many apoptotic stimuli. In this study, we have investigated the molecular mechanism in NPe6-PDT-induced apoptosis in human lung adenocarcinoma cells (ASTC-a-1). A recombinant fluorescence resonance energy transfer (FRET) Bid probe was utilized to determine the kinetics of Bid cleavage. The cells were also transfected with Bid-CFP to dynamically detect tBid translocation. The results show that cleavage of the Bid-FRET probe occurred 150±5 min after NPe6-PDT treatment, and this process lasted for 45±5 min. The Bid cleavage coincided with a translocation of tBid from cytoplasm to mitochondria. Using real-time single-cell analysis, we first observed the kinetics of Bid cleavage and translocation to mitochondria in living cells during NPe6-PDT-induced apoptosis