This chapter describes a system using adeno-associated viral (AAV) vector to deliver hypoxia-inducible gene expression to ischemic hearts. The hypoxia induction of gene expression in this system is based on the accumulation of hypoxia-inducible factor-1 (HIF-1) in ischemic hearts and the use of hypoxia-response element (HRE) identified from the enhancers of genes, the expression of which can be induced by hypoxia. The methods of plasmid and AAV vector construction for hypoxia-inducible gene expression, viral vector production and purification, and viral titer determination are described. This chapter also illustrates the methods that can be used to test hypoxia-inducible gene expression in vitro and in vivo, including hypoxia treatment of cultured cells, generation of murine ischemic heart models, and analysis of gene expression.