alpha(1)-Adrenergic signaling in cardiac myocytes activates the skeletal muscle alpha-actin gene through an MCAT cis-element, the binding site of the transcriptional enhancer factor-1 (TEF-1) family of transcription factors. TEF-1 accounts for more than 85% of the MCAT binding activity in neonatal rat cardiac myocytes. Other TEF-1 family members account for the rest. Although TEF-1 itself has little effect on the alpha(1)-adrenergic activation of skeletal muscle alpha-actin, the related factor RTEF-1 augments the response and is a target of alpha(1)-adrenergic signaling. Here, we examined another TEF-1 family member expressed in cardiac muscle, DTEF-1, and observed that it also augmented the alpha(1)-adrenergic response of skeletal muscle alpha-actin. A DTEF-1 peptide-specific antibody revealed that endogenous DTEF-1 accounts for up to 5% of the MCAT binding activity in neonatal rat cardiac myocytes. A TEF-1/DTEF-1 chimera suggests that alpha(1)-adrenergic signaling modulates DTEF-1 function. Orthophosphate labeling and immunoprecipitation of an epitope-tagged DTEF-1 showed that DTEF-1 is phosphorylated in vivo. alpha(1)-Adrenergic stimulation increased while phosphatase treatment lowered the MCAT binding by DTEF-1 and the endogenous non-TEF-1 MCAT-binding factor. In contrast, alpha(1)-adrenergic stimulation did not alter, and phosphatase treatment increased, MCAT binding of TEF-1 and RTEF-1. Taken together, these results suggest that DTEF-1 is a target for alpha(1)-adrenergic activation of the skeletal muscle alpha-actin gene in neonatal rat cardiac myocytes.