Ostium secundum atrial septal defect (osASD) is one of the most commonly occurring cardiac malformations. Although some embryological pathways have been elucidated, the molecular etiologies of ASD are not fully understood. Previous microarray analysis in our laboratory identified differentially expressed genes between osASD and normal right auricular myocardium. Of the 1056 differentially expressed genes, 14 genes were related to apoptosis: eight pro-apoptotic genes were up-regulated and six anti-apoptotic genes were down-regulated in ASD patients. In the current study, we utilized semi-quantitative RT-PCR, electron microscopy, TUNEL and flow cytometry to further understand the role of apoptosis in the atrium of osASD patients. RT-PCR results confirmed differential expression data from previous microarray studies. Additionally, while apoptosis was detected in the right auricular myocardium of all osASD patients, it was absent in controls. These data suggested apoptosis may play an important role in the pathogenesis of osASD or possibly occurs as a consequence of volume overload and hemodynamic changes in right atrium of osASD patients.