Reduced glutathione (GSH) is the most abundant antioxidant in the cell which has a central place in the control of vital cell processes including cell proliferation. Considering the growing evidence that shows the importance of GSH compartimentation and its role in numerous processes that occur in the nucleus, we have studied the possible relation between nuclear glutathione and cell proliferation. Our model included 3T3 fibroblasts treated with a.100 mM diethylmaleate (DEM) that depletes all cellular thiols in entire cell, including nuclear GSH b.100mM DEM and 1mM GSH ester that enters the cell and protects it efficiently from GSH depletion induced by DEM, c.10mM BSO that prevents the GSH synthesis in 24h affecting mostly cytosolic GSH. GSH distribution was studied by fluorescence determination of its CMFDA conjugate using confocal microscopy and cell cycle by flow cytometry. Cells treated with DEM showed significant loss in GSH cellular level within 3h after treatment, lower percentage of cells in S+G2/M phase, and lower nucleus/cytosol GSH ratio comparing to control and to cells treated with both DEM and GSH ester. BSO treatment, although it lowered cellular GSH level, did not affect its nuclear compartimentation or the cell proliferation. We believe that the start of active proliferation of 3T3 fibroblasts requires a reduced environment in the nucleus in the first 24h after plating, and that the level of GSH compartimentation regulates the rhythm of cell cycle.