Methanotrophs expressing soluble methane monooxygenase (sMMO) may find use in a variety of industrial applications. However, sMMO expression is strongly inhibited by copper, and the growth rate may be limited by the aqueous solubility of methane. In this study, addition of allylthiourea decreased intracellular copper in Methylosinus trichosporium OB3b, allowing sMMO production at Cu/biomass ratios normally not permitting sMMO synthesis. The presence of about 1.5 micromoles intracellular Cu g(-1) dry biomass resulted in sMMO activity of about 250 micromoles 1-napthol formed per hour gram dry biomass whether this intracellular Cu concentration was achieved by Cu limitation or by allylthiourea addition. No loss of sMMO activity occurred when the growth substrate was switched from methane to methanol when allylthiourea had been added to growth medium containing copper. Addition of copper to medium that was almost copper-free increased the yield of dry biomass from methanol from 0.20 to 0.36 g g(-1), demonstrating that some copper was necessary for good growth. This study demonstrated a method by which sMMO can be produced by M. trichosporium OB3b while growing on methanol in copper-containing medium.