The potential of a femto-injection technique for use in analyzing protein dynamics in embryonic stem (ES) cells was investigated. First, we showed that fluorescent proteins could be injected in a quantitative fashion into individual mouse ES cells. Second, we demonstrated that the technique could identify functional differences between proteins by analyzing the effect of a nuclear localization signal on the behavior of glutathione S-transferase conjugated to green fluorescent protein. The analysis showed a clear difference in the distribution of the protein when the nuclear localization signal was present. Our results confirm that the non-destructive, quantitative and time controllable aspects of the technique provide considerable advantages for the analysis of protein behavior in living ES cells. To the best of our knowledge, this is the first report of the successful introduction of proteins into living ES cells by an injection technique.