We have isolated and sequenced the gene and the cDNA coding for the human cardiac β-myosin heavy chain (designated MYH7). The gene is 22,883 bp long. The 1935 amino acids of this protein (M_r 223,111) are encoded by 38 exons. The 5′ untranslated region (86 bp) is split by two introns. The 3′ untranslated region is 114 bp long. Three Alu repeats were identified within the gene and a fourth one in the 3′ flanking intergenic region. The molecular organization of this gene reflects the conservative pattern with respect to size, coding ratio, and number or position of introns characteristic of vertebrate sarcomeric myosin heavy chain genes. The protein sequence of the human β-heavy chain was compared with corresponding (homologous) sequences of rabbit, rat, and hamster as well as with the (heterologous) embryonic heavy chain sequences of rat, chicken, and man. The results show that protein subregions responsible for basic functions of myosin heavy chains (nucleotide binding and actin binding) are very similar in homologous and heterologous heavy chains. Regions that differ in their primary sequences in heterologous heavy chains appear to be highly conserved within mammalian β-myosin heavy chains. Constant and variable subregions of heavy chains are discussed in terms of functional significance and evolutionary relatedness.