Cultures of Methylosinus trichosporium OB3b grown in the presence of very low concentrations of copper synthesize a soluble methane monooxygenase (sMMO) that efficiently catalyzes the oxidation of trichloroethylene and other organic pollutants. Recently, we isolated five M. trichosporium OB3b mutants that express sMMO activity when grown in the presence of elevated copper concentrations (P.A. Phelps, S. K. Agarwal, G. E. Speitel, Jr., and G. Georgiou, Appl. Environ. Microbiol. 58:3701-3708, 1992). Here we show that, in contrast to the results for the wild-type cells, the addition of copper to mutant cultures grown on methane and nitrate as the nitrogen source has no noticeable effect on the growth rate and sMMO expression. In vitro experiments indicated that the copper-resistant phenotype does not arise from an increased stability of sMMO to copper deactivation. Furthermore, the mutant cultures exhibit altered speciation of copper in the extracellular fluid and have substantially decreased levels of cell-associated copper. On the basis of these results, we propose that the mutant phenotype arises from defects in copper uptake and metabolism rather than from changes in sMMO expression or enzyme stability.