A new restricted facultative methylotroph, strain LTKT, isolated from the water of a small freshwater reservoir, was found to carry out the KDPG variant of the ribulose monophosphate pathway of С1 meta-bolism. The cells of the isolate were aerobic, gram-negative, non-spore-forming, nonmotile rods reproducing by binary fission. The isolate was mesophilic and neutrophilic; it synthesized indole derivatives. Hexadecanoic (C16:0) and cis-9-hexadecenoic (C16:1ω7) acids were the major fatty acids, and Q8 was the major ubiquinone. Phosphatidylethanolamine and phosphatidylglycerol were the dominant phospholipids, while diphosphatidylglycerol was not present. Activities of α-ketoglutarate dehydrogenase and glutamate dehydrogenase were not revealed. Ammonium was assimilated via the enzyme system of the glutamate cycle. The 16S rRNA gene sequence of strain LTKT exhibited high similarity to those of Methylophilus species: 99.6% to M. leisingeri DM11T and 99.4% to M. flavus ShipT. DNA‒DNA homology between strain LTKT and M. leisingeri DM11T and M. flavus ShipT was 47 and 40%, respectively. Based on these data, strain LTKT was assigned to the new species, Methylophilus aquaticus sp. nov. (= VKM B-3159T = JCM 32032T = CCUG 70602T).