A soluble methane monooxygenase (sMMO: EC 1.14.13.25) was purified from a type II obligate methanotroph, Methylocystis sp. M. Ion exchange chromatography elution separated the sMMO into three components, I, II, and III. Components II and III were purified to homogeneity and were essential for the sMMO activity. Components II and III had molecular masses of approximately 233,000 and 39,000, respectively. Component II consisted of three subunits with molecular masses of 55,000, 44,000, and 21,000, which appeared to be present in stoichiometric amounts, suggesting a (αβγ)2 configuration in the native protein. Component II contained 1-4 mol of iron and was considered to be a hydroxylase. Component III was a flavoprotein, which contained 1 mol of FAD as well as 1-2mol of iron. It catalyzed the reduction of K3Fe(CN)6 and 2,6-dichloroindophenol by NADH. Component I, which was partially purified and not essential for sMMO activity, stimulated the activity by about 11-fold. Its stimulation could be replaced by addition of Fe(2+). The molecular mass of the partially purified component I was estimated to be from 35,000 to 40,000 based on gel filtration, which suggested the presence of a new type of regulatory protein of sMMO.