Na-K-ATPase alpha 3-subunit mRNA levels are both positively and negatively controlled by thyroid hormone [3,5,3'triiodothyronine (T3)] in primary cultures of neonatal rat cardiac myocytes. In this study, transient transfection analysis indicated that two regions of the rat alpha 3 gene between nucleotides -116 and -6 and -6 and +80 conferred T3-mediated inhibition of reporter gene expression. Electrophoretic mobility shift assays showed specific binding of T3 receptor monomers and T3 receptor-retinoid X receptor heterodimers at each alpha 3 gene negative T3-response region. The alpha 3 gene region from -116 to -6 base pairs also mediates repression in response to retinoic acid (RA) and binds RA receptor. In the absence of ligand, reporter gene expression driven by the -116 to -6-base pair region is repressed with cotransfection of T3 receptor, whereas it is unaffected by overexpression of RA receptor. These data demonstrate that the proximal promoter of the rat Na-K-ATPase alpha 3 gene contains sequence motifs that mediate repression of alpha 3 gene transcription in response to either T3 or RA in neonatal rat cardiac myocytes.