We have obtained the 5820 nucleotide sequence encoding all 1939 amino acids of the human cardiac alpha-myosin heavy chain (alpha-MHC), as established by dideoxy sequencing of cloned cDNA, genomic DNA and polymerase chain reaction (PCR) amplification products. This sequence represents overlapping fragments of the entire coding sequence. Amino acid sequence comparison of the human cardiac alpha-MHC with the published human cardiac beta-MHC have demonstrated that there are, at least, 7 isoform-specific divergent regions, including functionally important binding protein-related sites such as ATP, actin and myosin light chain. It has been reported that in the rat, there are 8 isoform-specific divergent regions. The 7th divergent area (residue area 1633-1657, which is thought to mediate thick filament formation) in the light meromyosin region in the rat is not apparent in the human. The amino acid compositions of cardiac alpha- and beta-MHCs in the human and the rat, and human embryonic skeletal muscle and chicken gizzard smooth muscles were compared. Amino acid sequences in cardiac alpha- and beta-MHCs in the human and the rat are well conserved. In the head portion, the amino acid composition divergence of human cardiac alpha-MHC is ranked between rat cardiac alpha-MHC and human cardiac beta- or rat cardiac beta-MHC; human skeletal muscle MHC is the most divergent of the myosin isoform examined. These data predict that human cardiac alpha-MHC may have undergone evolutionary changes toward obtaining the biochemical and physiological properties of cardiac beta-MHC.