Cardiac chamber specification is a complex process orchestrated by multiple signaling pathways and morphogenetic movements. It has been demonstrated that Chicken Ovalbumin Promoter Transcription Factor II (COUPTF-II) and the Slow Myosin Heavy Chain III (SMyHC III) genes are selectively expressed in atria of embryonic hearts. More recently, we have established that the SMyHC III promoter contains a complex Nuclear Receptor Element (cNRE) that is capable of being recognized by a wide array of transcription factors of the Nuclear Receptor Superfamily, including COUPTF-II and Androgen Receptor (AR). Thus, we sought to determine whether COUPTF-II binds to the cNRE to drive the atrium-specific expression of the SMyHC III gene. We performed a Nuclear Receptor transactivation screening in HEK293-T cells expressing reporter constructs harboring wild-type and mutant SMyHC III promoter for the cNRE, as well as heterologous fusions of the cNRE (1X or 5X) to the basal SV40 promoter to gain insights on the roles of COUPTF-II played via the cNRE. We observed that either COUPTF-II or AR represses the activity of the SMyHC III promoter. However, upon co-transfection, COUPTF-II and AR activate the SMyHC III promoter. The activating effect of COUPTF-II plus AR is not observed in the context of a cNRE-less promoter, indicating that this sequence is necessary for stimulation. Surprisingly, the activating behaviour of the COUPTF-II-AR combination is not expressed in the heterologous context of cNRE-SV40 constructs, indicating that the cNRE, though necessary, is not sufficient. We demonstrated the participation of AR as a novel player in cardiac chamber specification, and its synergic interaction with COUPTF-II to promote the activation of the atrial-specific gene program. In conclusion, our data unveils new roles for known transcription factors and reveals new mechanisms underlying cardiac chamber specification.