Objective: The right ventricle (RV) demonstrates differential adaptations in response to pressure versus volume loading, a phenomenon that may be important in the management of children and adults with congenital heart disease (CHD). The purpose of this study is to elucidate possible transcriptional mechanisms of the RV response to pressure versus volume loading in vivo. Methods: Fetal lambs had aortopulmonary shunting or pulmonary artery (PA) banding. Four weeks after spontaneous delivery, ovine hearts were evaluated for hemodynamic changes and changes in expression of sarcomeric gene proteins and transcriptional factors. Western blot densitometry and chromatin immunoprecipitation were applied using standard techniques. Transactivation assays were performed using transient transfections in Schneider's Drosophila line 2 cells in culture. Results: After PA banding, the RV pressure increased from 36 ± 4 mm Hg (n = 4) to 96 ± 8 mm Hg (n = 4, P < .05). The RVs of shunted and banded animals showed significant increases in the expression levels and promoter binding of activators myocyte enhancer factor 2, GATA-4, Nkx2.5, transcriptional enhancer factor 1, and specificity protein (Sp) 1. The transcriptional repressor Sp3 was downregulated in shunted animals, but its expression was increased paradoxically in the RV of the PA band group. Immunoprecipitation of Sp3 showed posttranslational modification to the acetylated isoform. In transient transfections of Schneider's Drosophila line 2 cells, acetylation of Sp3 converted it from a transcriptional repressor to an activator. Conclusions: Posttranslational modifications of the transcriptional repressor Sp3, by acetylation, may be an important mechanism in the differential response of the RV to abnormal loading conditions. Sp3 may serve as a biomarker for RV failure for various heart defects in children and adults with CHD. These findings may have therapeutic implications in the management of right heart failure.