Peroxisome proliferation activator receptor (PPAR) gamma-coactivator 1alpha (PGC-1alpha), a transcription coactivator, functions as a master regulator of a wide array of metabolic and physiological processes and is an essential factor in the process of mitochondrial biogenesis. Transfection of NIH 3T3 fibroblasts with a mouse cDNA for PGC-1alpha led to the induction of markers of mitochondrial biogenesis, that is, mitochondrial transcription factor A (mtTFA), cytochrome c, and mitochondrial DNA (mtDNA). Mitochondrial biogenesis-associated net protein synthesis appears to be accomplished by a reduction in the rate of mitochondrial protein degradation with little or no change in the rate of protein synthesis. Overexpression of PGC-1alpha did not adversely affect cellular proliferation. Cellular ATP levels were increased in the transfected cells and they were more resistant to oxidative stress than the control nontransfected 3T3 cells. This resistance to oxidative stress was manifested by both an improved viability and the maintenance of mitochondrial membrane potential in the transfected cells when exposed to t-butyl hydroperoxide (t-BOOH). It therefore appears that PGC-1alpha overexpression stimulates mitochondrial biogenesis in 3T3 cells making them more resistant to oxidative stressors.