Although several studies have suggested relatively slow turnover of Langerhans cells (LCs), their actual lifespan remains elusive. Here we report the development of a new intravital imaging system for studying LC efflux and influx. Epidermal LCs expressing enhanced green fluorescent protein (EGFP) were visualized in anesthetized I-Abeta-EGFP knock-in mice by confocal microscopy. By overlaying two sets of EGFP+ LC images recorded in the same microscopic fields at time 0 and 24 hours later, we identified LC subpopulations that had disappeared from or newly emerged in the epidermis during that period. Of >10,000 LCs analyzed in this manner, an overwhelming majority (97.8+/-0.2%) of LCs showed no significant changes in the x-y locations, whereas 1.3+/-0.1% of the LCs that were found at time 0 became undetectable 24 hours later, representing LC efflux. Conversely, 0.9+/-0.1% of the LCs that were found at time 24 hours were not detectable at time 0, representing LC influx. From these frequencies, we estimated the half-life of epidermal LCs to range from 53 to 78 days, providing new insights into the immunobiology of LCs. Our intermittent imaging approach may be regarded as a technical breakthrough enabling direct visual assessment of LC turnover in living animals.