The structures of methanol dehydrogenase (MEDH) from two closely related methylotrophic bacteria, Methylophilus methylotrophus and W3A1, have been determined at 2.6-A resolution. The molecule, a quinoprotein of molecular mass of about 138 kDa, contains two heavy (H) and two light (L) subunits of unknown sequence and two molecules of noncovalently associated pyrroloquinoline quinone. The two enzymes crystallize isomorphously in space group P2(1) with one H2L2 heterotetramer in the asymmetric unit. The electron density map of the M. methylophilus enzyme was obtained by multiple isomorphous replacement with anomalous scattering and improved by solvent leveling and electron density averaging. For model building, the amino acid sequence of MEDH from Paracoccus denitrificans for the H subunit and from Methylobacterium extorquens AM1 for the L subunit were used to represent the unknown amino acid sequence. At the present time, 579 and 57 amino acid residues for the large and small subunits, respectively, have been fitted into the map. The phases for MEDH from M. methylophilus were used directly to analyze the W3A1 structure, and both structures were refined to R-factors (where R = sigma[Fo-Fc[/sigma Fo) of 0.277 and 0.266, respectively. The L subunit contains a long alpha-helix and an extended N-terminal segment, both lying on the molecular surface of the H subunit. The H subunit contains eight antiparallel beta-sheets, each consisting of four strands arranged topologically like the letter W. The eight Ws are arranged circularly, forming the main disc-shaped body of the subunit, with some short helices and loops connecting the consecutive Ws, as well as some excursions within and between some of the Ws. The pyrroloquinoline quinone prosthetic group is located in the central channel of the large subunit near the surface of the molecule. The topology of the eight-W folding unit is similar to those of the six- and seven-W folding units previously reported for three other proteins, neuraminidase, methylamine dehydrogenase, and galactose oxidase.