Aims: Anesthetic preconditioning (APC), defined as brief exposure to inhalational anesthetics prior to cardiac ischemia/reperfusion (I/R), limits injury in both animal models and in humans. APC can result in production of reactive oxygen species, and prior work has shown that APC can modify activation NF-B during I/R, with consequent reduction in the expression of inflammatory mediators. However, the role of NF-B activation prior to I/R is unknown. Therefore, these experiments tested the hypothesis that APC-induced ROS results in activation of NF-B prior to I/R, with consequent increased expression of anti-apoptotic proteins such as Bcl-2 and decreased apoptosis. Methods: Experiments utilized an established perfused heart rat model of APC and I/R. The role of NF-B was defined by a novel method of transient inhibition of the regulatory kinase IKK using the reversible inhibitor SC-514. In addition to functional measures, phosphorylation of IB and activation of NF-B were measured along with cytosolic protein content of Bcl-2, release of cytochrome c, and degradation of caspase-3. Results: APC resulted in ROS-dependent phosphorylation of IB and activation of NF-B prior to I/R. APC also increased expression of Bcl-2 prior to I/R. In addition to functional protection following I/R, APC resulted in lower release of cytochrome c and caspase-3 degradation. These protective effects of APC were abolished by transient inhibition of IB phosphorylation and NF-B activation by SC-514 followed by washout. Conclusions: ROS-dependent activation of NF-B by APC prior to I/R is a critical element in the protective effect of APC. APC reduces apoptosis and functional impairment by increasing Bcl-2 expression prior to I/R. Interventions which increase NF-B activation prior to I/R should protect hearts from I/R injury.