Two genes encoding cardiac MHC isoforms, and , are arranged in tandem 4.5 Kb apart. We examined pre-mRNA and mature mRNA levels of and in control, diabetic (STZ), hypothyroid (PTU), and hyperthyroid (T3) rat hearts. We also analyzed the naturally occurring antisense (AS) RNA species that starts in the middle of the 4.5Kb intergenic region and extends upstream to the gene promoter. The and genes are expressed in an antithetical manner in control, diabetic, hypothyroid, and hyperthyroid hearts. The expression of the AS RNA was positively correlated with the mRNA and negatively correlated with the sense mRNA. These RNA results support the novel idea that there are common promoter regulatory elements situated in the intergenic region that likely control transcription of both the sense and the AS genes and that the AS transcription negatively regulates MHC gene expression. To test whether an intergenic promoter drives the transcription of the AS RNA, a 1340bp sequence of the intergenic region was inserted into a luciferase plasmid in the 3 to 5 AS direction, and was injected into rat ventricle. This promoter was activated in control heart and decreased greatly in response to PTU and STZ and increased in hyperthyroid rats, similar in pattern to the endogenous AS RNA. When a putative retinoic acid receptor (RAR) site (a known thyroid hormone receptor cofactor) in this promoter was mutated, the reporter activity was almost abolished in Cont, PTU, and STZ hearts. We conclude that there is an intergenic promoter that is active in the AS direction and that the putative RAR element is a vital regulatory site.