Digital electron images of frozen-hydrated preparations of the 2.25-MDa Staphylothermus marinus phosphoenolpyruvate synthase (EC 2.7.9.2) have been analyzed by single-particle classification and averaging and iterative quaternion-based angular reconstitution. Contrast transfer function correction of micrographs obtained at different defocus values was used to improve the informational quality of the projection averages. Three-dimensional reconstructions were obtained to roughly 3-nm spatial resolution, in which the 24 identical subunits were,arranged to form an octahedral complex, although:the amino-terminal nucleoticle-binding domain was not resolved. An atomic model of the subunit was generated by homology modeling using as the reference the known X-ray crystallographic structure of the related enzyme pyruvate orthophosphate dikinase (EC 2.7.9.1) from Clostridium symbiosum (Protein Data Bank entry 1DIK). The S. marinus protein could be arranged into an assembly of 12 homodimers to match the three-dimensional reconstruction in terms of shape and size of the homodimers, as well as overall shape and size of the:complex. The quaternary model indicated that active sites of three monomers were localized around cavities (or putative channels) centered at the threefold axes of rotational symmetry and that carboxyl-terminal alpha-helical segments of four monomers were localized at the fourfold axes of rotational symmetry where they could facilitate interdimer interaction, The quaternary arrangement also indicated numerous potential hydrophobic and electrostatic interactions at the interdimer interfaces that could contribute further to structural stability,