Publisher Summary This chapter focuses on oxygen-regulated signal transduction pathways in the heart. Cellular O2 concentrations are maintained within a narrow range (normoxia) due to the risk of oxidative damage from excess O2 (hyperoxia) and of metabolic demise from insufficient O2 (hypoxia). The pO2 ranges from 90 to below 3 Torr in mammalian organs under normoxic conditions with an arterial pO2 of about 100 Torr or ∼14% O2. The normoxia for cells is a variable that is dependent on the specific localization of the cell in organs and functional status of the specific tissue. O2 sensing is required to adjust to physiological or pathophysiological variations in pO2. In this chapter, concepts related to oxygenation of the heart are discussed, along withcellular responses to reoxygenation following chronic moderate hypoxia and the experimental setup developed to control oxygen levels during all procedures of cell culture as well as while performing live cell imaging. Details of cell culture and live cell imaging under controlled O2 environment and oxygen-regulated signal transduction pathways in cardiac cells are alsodescribed.