We have used the specific reaction of monochlorobimane (mBCI) with GSH to analyze hepatic GSH, mBCI, itself nonfluorescent, forms a stable, fluorescent adduct with GSH in a reaction catalyzed by the GSH S-transferases (GST). When hepatocytes were labeled with mBC1 (100 microM) in Krebs-Henseleit buffer, the fluorescent signal recorded over time was directly proportional to the concentration of GSH. The HPLC analyses of hepatocytes that were preloaded with the dye indicated that GSH was the only thiol labeled. When the technique was applied to freshly isolated intact hepatocytes that contained different levels of GSH, a close correlation between the levels of GSH measured by the present method (mBC1) and the standard enzymatic recycling method was found. A similar agreement for the cytosolic and mitochondrial pools of GSH determined by the two methods was established. The fluorescent GSH-bimane adduct, once formed within the cell, was not released from the cell. In addition, we have applied this technique to determine directly the rate of synthesis of GSH in both cell-free conditions and in cell suspensions by monitoring the increase in fluorescent adduct when mBC1 is present in excess in the incubation.