Soluble methane monooxygenase (sMMO : EC 1.14.13.25) from Methylocystis sp. M is a multicomponent enzyme consisting of a hydroxylase, a reductase, and component B. The hydroxylase and the reductase have been purified and characterized (Nakajima, Uchiyama, Yagi, and Nakahara, 1992). We describe a purification protocol for the uncharacterized component B. Component B has a molecular mass of approximately 32,000, consisting of 2 subunits, each with a molecular mass of 15,100. It contains neither metals nor prosthetic groups. The protein was gradually truncated from the N-terminal end and lost 30 amino acid residues, forming components B＇ or B " with molecular masses of 11,100 and 10,500, respectively. At the truncation site, the secondary structure of component B changed from alpha-helix to beta-sheet. Component B appeared to have a higher affinity for the hydroxylase than the reductase. It also increased the heat stability of the hydroxylase and retarded separation of iron from the active center of the hydroxylase. From these results, it is suggested that the role of component B in the sMMO system is the stabilization of the hydroxylase structure rather than electron transfer from reductase to hydroxylase.