Abstract Congenital heart defects constitute a significant pediatric and adult health problem. They not only represent the leading non-infectious cause of death in infants, but the growing number of adult survivors also suffer significant morbidity. Numerous genetic contributors have been identified to cause congenital heart defects ranging from chromosome abnormalities to single gene defects. Congenital heart disease-causing genes have been well-studied using animal models and the molecular pathways regulating normal cardiac morphogenesis are being increasingly defined. Environmental factors are known to increase the incidence of congenital heart defects. A significant knowledge gap exists in our understanding of the mechanisms by which environmental factors affect the molecular pathways regulating heart development to increase the risk of congenital heart disease. Maternal diabetes is a well-established and increasingly prevalent environmental risk factor for congenital heart disease. We recently described a novel gene-environment interaction between maternal hyperglycemia and Notch1 signaling that increased the risk of congenital heart defects in animal models. Our studies suggested that alterations in the epigenetic regulation of the nitric oxide and Notch1 signaling pathways were responsible for this interaction. Our long-term objective is to define the molecular and cellular pathways underlying this interaction and define a paradigm by which gene-environment interactions occur to cause congenital heart defects. The overall hypothesis of this research is that maternal diabetes-associated congenital heart disease occurs when maternal hyperglycemia-induced reactive oxygen species disrupts normal cardiac morphogenesis by epigenetic mechanisms and will be addressed in the following specific aims: Aim 1. To determine the cell-specific and temporal mechanisms by which hyperglycemia-associated oxidative stress mediates reduced chromatin accessibility at the Nos3 locus to cause congenital heart disease. Aim 2. To define the mechanism by which nitric oxide regulates the expression of the epigenetic Notch1 regulator, Jarid2. Aim 3. To determine if alterations in chromatin regulatory genes that occur with hyperglycemia contribute to maternal diabetes-associated congenital heart defects. Elucidating the molecular basis for the epidemiologic association between diabetes and cardiac malformations is required in order to devise novel preventive strategies for diabetes-associated congenital heart disease and potentially identify at risk individuals. Successful completion of the proposed studies will result in a significant advancement in our molecular understanding of the mechanisms by which gene-environment interactions contribute to congenital heart defect occurrence.