Combinatorial interactions between cis-elements and trans-acting factors are required for the regulation of cardiac gene expression during normal cardiac development and pathologic cardiac hypertrophy. The Sp family of transcription factors bind GC-boxes and are implicated in the recruitment and assembly of the basal transcriptional complex. In this study we show that the cardiac troponin T promoter contains a GC box that is necessary for basal and cAMP-mediated activity of cTnT promoter constructs transfected in embryonic cardiomyocytes. Cardiac nuclear proteins bind the cTnT GC-box in a sequence-specific fashion and are comprised of Sp1, 2 and 3 protein factors. By chromatin immunoprecipitation, Sp1 binds the cTnT promoter, "in vivo". Cotransfected Sp1 transactivates the cTnT promoter in primary cardiac myocytes in culture. Sp3 represses Sp1-mediated transcriptional activation of cTnT promoter constructs. Sp3 repression is dose-dependent inferring a mechanism of competitive binding/inhibition. In order to evaluate the role of Sp factors in normal and pathologic cardiac gene expression, in vivo, we have established a clinically-relevant animal model of pathologic cardiac hypertrophy where the fetal cardiac program is activated. In this animal model, cardiac hypertrophy results from increased left-right shunting, volume loading of the left ventricle and pressure loading of the right ventricle. Sp1 expression is increased in all 4 hypertrophied cardiac chambers while that of Sp3 is diminished. The observation is consistent with the in vitro activating function that Sp1 and inhibitory effects that Sp3 have on activity of cTnT promoter constructs. Sp factor levels are modulated during the hypertrophic cardiac program, in vivo.