MCAT elements are essential for cardiac gene expression during development. Avian TEF-1 proteins are muscle-enriched and contribute to MCAT binding activities. However, direct activation of MCAT-driven promoters by TEF1-related proteins has not been uniformly achieved. DTEF-1 is a unique member of the TEF-1 multigene family with abundant transcripts in heart but not skeletal muscle. Here we show that DTEF-1 proteins are highly expressed in heart. Protein expression is activated at very early stages of chick embryogenesis (Hamburger- Hamilton HHstage 4, 16-18hours) after which DTEF-1 becomes abundant in the sinus venosus and is expressed at lower levels in the trabeculated, ventricular myocardium and ventricular outflow tracts. By chromatin immunoprecipitation, DTEF-1 interacts with the cTnT promoter in vivo. DTEF-1 also interacts with MEF 2 by co-immunoprecipitation and independently or co-operatively (with MEF 2) trans-activates the cTnT promoter. DTEF-1 isoforms do not activate the cTnT promoter in fibroblasts or skeletal muscle. DTEF-1 expression occurs very early in chick embryogenesis (16-18 hours), preceding sarcomeric protein expression, and it activates cardiac promoters. As such, DTEF-1 may be an early marker of the myocardial phenotype. DTEF-1 trans-activates the cTnT promoter in a tissue-specific fashion independent of AT-rich, MEF-2 or GATA sites. The observed spatial pattern suggests decreasing levels of expression from the cardiac inlet to the ventricular outflow tracts which may mark a cardiogenic or differentiation pathway that parallels the direction of flow through the developing chick heart.