Using photon counting and charge-coupled device (CCD) cameras, we have applied the method of real-time bioluminescence imaging to investigate protein trafficking in mammalian cells. In the living cells of Chinese hamster ovary and PC12D cells, exocytotic secretion of protein and protein targeting on the cell surface were visualized using the secreted Gaussia luciferase (GLase) as a reporter protein in a minute. After incubation of the cells with luciferin (coelenterazine) for 10 min, luciferin was imported into the cells and the vesicle transport network in the cells could be shown by luminescence images of GLase activity. Further, we demonstrate that GLase with a heterologous signal peptide sequence is targeted to the cell surface in neuronally differentiated PC12D cells and luminescence signals could be detected in a few seconds.