The majority of mitochondrial proteins are encoded by nuclear genes and synthesized in the cytosol as preproteins containing a mitochondria import sequence. Preproteins traverse the outer mitochondrial membrane in an unfolded state, then translocate through the inner membrane into the matrix via import machinery that includes mitochondrial heat shock protein 70 (mthsp70). Neonatal rat cardiac myocytes (NCM) infected with an adenoviral vector expressing mthsp70 or an empty control (Adv-) for 48 hours were submitted to 8 hours of simulated ischemia (hypoxia) followed by 16 hours of reperfusion (reoxygenation). Infection with mthsp70 virus yielded an increase in mthsp70 protein in NCM mitochondria as compared to Adv- (P<0.05). Cell viability following simulated I/R was decreased in both Adv- and mthsp70 groups, relative to control (P<0.05), but mthsp70-infected NCM had enhanced viability following I/R relative to Adv-infected NCM (P<0.05). Simulated I/R caused an increase in reactive oxygen species (ROS) generation and lipid peroxidation in Adv-infected NCM (P<0.05, for both), which was not observed in mthsp70-infected NCM. Mitochondrial complex III and IV activities were greater in mthsp70-infected NCM following simulated I/R as compared to Adv- (P<0.05 for both). Following simulated I/R, ATP content increased in mthsp70-infected NCM, as compared to Adv- (P<0.05). Apoptotic markers were decreased in mthsp70-infected NCM as compared to Adv- after simulated I/R (P<0.05). These results indicate that overexpression of mthsp70 protects the mitochondria against damage from simulated I/R, which may be due to a decrease in ROS leading to preservation of mitochondrial complex function activities and ATP formation.