Selected new fluorogenic probes that interact in different ways with Hg2+ and MeHg+ have been prepared and used for the chemical speciation of both cations in aqueous solution as well as in HEK293 cells. The best selective speciation of Hg2+ and MeHg+ has been achieved by in vitro approaches based on fluorogenic probes supported in cultured cells, due to the particular sensitivity of the HEK293 cells to permeation by Hg2+, MeHg+ and the fluorogenic probes. In particular, MeHg+ was selectively detected in cell nuclei by probe JG45.