Ventricular septal defect (VSD) accounts for the largest number of birth congenital heart defects in human, but the genetic programs that control ventricular septation are poorly understood. To identify differentially expressed genes between ventricular septal defect and normal ventricular septum myocardium, we have undertaken suppression subtractive hybridization (SSH) and generated reciprocal cDNA collections of representative mRNAs specific to human heart with ventricular septal defect versus normal control. Following SSH, 1378 clones were sequenced and found to derive from 551 different genes. These predominately expressed genes included genes involved in energy metabolism, cell cycle and growth, cytoskeleton and cell adhesion, LIM protein, zinc finger protein, and development. It is anticipated that further study of genes identified will provide insights into their specific roles in the etiology of VSD, even in cardiac development, aging, and disease.