Dynamics of excited state proton transfer (ESPT) in the lysosome region of live lung cells (normal and cancer) is studied by picosecond time-resolved confocal microscopy. For this, we used a fluorescent probe, pyranine (8-hydroxy-pyrene-1,3,6-trisulfonate, HPTS). From the colocalization of HPTS with a lysotracker dye (lysotracker yellow), we confirmed that HPTS resides in the lysosome for both of the cells. The diffusion coefficient (Dt) in the lysosome region was obtained from fluorescence correlation spectroscopy (FCS). From Dt, the viscosity of lysosome is estimated to be 40 and 30 cP in the cancer and normal cells, respectively. The rate constants of the elementary steps of ESPT in a normal lung cell (WI38) are compared with those in a lung cancer cell (A549). It is observed that the time constant of the initial proton transfer process in a normal cell (τPT = 40 ps) is similar to that in a cancer cell. The recombination of the geminate ion pair is slightly faster (τrec = 25 ps) in the normal cell than that (τrec = 30 ps) in a cancer cell. The time constant of the dissociation (τdiss) of the geminate ion pair for the cancer cell (τdiss = 80 ps) is 1.5 times faster compared to that (τdiss = 120 ps) in a normal cell. © 2014 American Chemical Society.