An electrophoretic comparison of 15 enzymes was made on 93 strains of gram-negative methanol-utilizing bacteria. Phenazine methosulfate (PMS)-linked methanol dehydrogenase, phosphoglucomutase, glucose-6-phosphate dehydrogenase (NADP-dependent), isocitrate dehydrogenase (NADP-dependent) and catalase were detected in almost all of the bacteria tested. PMS-linked methanol dehydrogenase was divided into 2 major types on the basis of the electrophoretic patterns. The 1st type of the enzyme was characterized by not migrating into gel at pH 8.9, and was found in the so-called obligate methanol-utilizing bacteria (group 1). The 2nd type of the enzyme moved into the gel during electrophoresis, and was found in Pseudomonas extorquens and related bacteria (group 2), Microcyclus strains (group 3), and Hyphomicrobium strains (group 4). The presence of hexose phosphate synthetase and the absence of .beta.-hydroxypyruvate reductase suggest the presence of the allulose pathway of methanol utilization in the bacteria of group 1; the absence of hexose phosphate synthetase and the presence of .beta.-hydroxypyruvate reductase suggests the presence of the serine pathway of methanol utilization in the bacteria of groups 2 and 4. Esterases were detected in the bacteria of groups 2, 3 and 4, but not in the bacteria of group 1. A considerable number of strains showed the same patterns in esterases, and such strains exhibited identical electrophoretic patterns in all other enzymes tested. Dendrograms were drawn on the basis of kinds of enzymes and their relative mobilities, and the clustering in each of the groups showed a good correlation with the previous grouping on the basis of the cellular fatty acid composition, the coenzyme Q system, and the phenotypic characteristics. [Group 1 bacteria included Methanomonas methylovora, M. methylovora ssp. thiaminophagus, Methylomonas methanolica, Protaminobacter thiaminophagus, Pseudomonas methylotropha and Pseudomonas sp. Group 2 bacteria included Protaminobacter sp., P. ruber, Pseudomonas sp., P. rosea and P. extorquens. Group 3 bacteria included Microcyclus aquaticus, M. eburneus and M. polymorphum. Group 4 bacteria included Hyphomicrobium sp. and H. variabile.].