The N-terminal amino acid sequences of six hemocyanin subunits from the Asian horseshoe crab Carcinoscorpius rotundicauda were analyzed and compared with those of eight hemocyanin subunits from the American horseshoe crab Limulus polyphemus. From common sequence traits, it is suggested that the two subunit sets, HR2-LP2A-LP3A and HR4-LP5-LP6, have originated respectively from a corresponding subunit in their common ancestor. From the N-terminal sequence comparison among four horseshoe crabs, it is inferred that the common ancestor of four horseshoe crabs had six hemocyanin subunits and that, after the divergence of two lineages of L. polyphemus and Asian horseshoe crabs, duplications of two subunits gave rise to two subunits sets (LP2A-LP3A and LP5-LP6) in the lineage of L. polyphemus and gave the eight different subunits to L. polyphemus. Thus, it is evident that the four extant horseshoe crabs share six orthologous hemocyanin subunits derived from their common ancestor.