Two ventricular myosin heavy chains (MHCs), alpha and beta, which exhibit different levels of ATPase activity, are differentially expressed during development, in response to thyroid hormone and in several pathological conditions. We have isolated and analyzed the structure of the genes coding for alpha- and beta-MHC mRNAs in the rat. Detailed analysis of eight overlapping MHC genomic clones shows that the alpha- and beta-MHC genes are organized in tandem and span 50 kilobases of the chromosome. The beta-MHC gene, predominantly expressed in late fetal life, is located 4 kilobases upstream from the alpha-MHC gene, predominantly expressed in the adult. These two genes are very closely related at the nucleotide sequence level, suggesting that they have arisen by duplication of a common ancestor, yet their expression in the ventricular myocardium has been shown to be regulated in an antithetic fashion by thyroid hormone.