Hemocyanins, the respiratory molecules of cephalopod mollusks, are hollow cylinders with five internal arches. Three hemocyanins representative of three orders of cephalopods (Benthoctopus species, Octopoda; Vampyroteuthis infernalis, Vampyromorpha; Sepia officinalis, Sepioidea) were subjected to cryoelectron microscopy and three-dimensional (3D) reconstruction. The structure of Benthoctopus hemocyanin, solved at 26.4-Angstrom resolution, possesses arches comprising two identical functional units. The similarity between these functional units and the structure recently observed in X-ray crystallography for Octopus by Cuff ct al. (J. Mol. Biol., 1998, 232, 522-529) allows the identification of their N- and C-terminal domains in this 3D reconstruction volume. Conversely, arches present in the 3D reconstruction volume of Sepia hemocyanin (21.8 Angstrom resolution) contain four functional units that are disposed differently. The strong resemblance between the reconstruction volumes of Vampyroteuthis (21.4-Angstrom resolution) and Benthoctopus hemocyanins suggests that Sepioidea diverged from a group containing Octopoda and Vampyromorpha.