The potential for performing dual analysis of intracellular glutathione levels and assessment of gap junctional intercellular communication with thiol-specific fluorescent probes in anchored cells was evaluated. Gap junction-mediated diffusion of monochlorobimane and 5-chloromethylfluorescein diacetate following intracellular loading and conjugation with glutathione was compared with 5-carboxyfluorescein diacetate (which is routinely used in laser cytometry to monitor intercellular communication) by means of fluorescence recovery after photobleaching using a variety of communication-competent and communication-incompetent cells. The rate of diffusion of fluorescence among communication-competent cells was inversely proportional to the size of the fluorescent probe employed. The thiol-specific probes were also employed to monitor depletion and synthesis of glutathione following treatments to inhibit glutathione synthesis or consume glutathione by adduct formation. Analysis of gap junctional intercellular communication following glutathione depletion revealed a direct correlation between glutathione levels and intercellular communication. These studies support the utility of the thiol-specific probes to monitor the respective role of cellular glutathione and intercellular communication in the mechanisms of cellular injury.