We generated cells with increased activities of GR by transfection of CHO cells with the cDNA for human GR, with and without a mitochondrial targeting signal (MTS). Clones G20 and GL13 showed higher GR activities (G20, 164 mU/mg pro; GL13, 184) than did CHO cells (28). GL13 cells, transfected with hGR with MTS cDNA had higher mitochondrial GR activities (434) than did G20 cells transfected with only hGR cDNA (56) or CHO cells (48). GSH concentrations were increased in both transfected cell lines, while GSSG concentrations did not change. Previous studies revealed that overexpression of GR in mitochondria is more protective against t-butyl hydroperoxide than extramitochondrial GR, but the effects of oxidants added extracellularly might not give an accurate assessment of protection against oxidants generated intracellularly, which we investigated in the present studies. Incubation of CHO and G20 cells with 0.1mM diquat caused increased release of LDH to the media beginning at 50 h and half-maximal release was observed at 60 h. In GL13 cells the effects of diquat were delayed by 20 h, and these cells showed little LDH leakage when the CHO and G20 cells showed almost complete cell death. These studies show that enhanced GR activities in mitochondria protect against the cytotoxic effects of reactive oxygen species generated intracellularly. Supported by GM44263 and a summer research fellowship (to VRR) by the Society for Pediatric Research.