Gene expression in methanotrophs has been shown to be affected by the availability of a variety of metals, most notably copper regulating expression of alternative forms of methane monooxygenase. Here, we show that growth substrate also affects expression of genes encoding for enzymes responsible for the oxidation of methane to formaldehyde and the assimilation of carbon. Specifically, in Methylosinus trichosporium OB3b, expression of genes involved in the conversion of methane to methanol (pmoA and mmoX) and methanol to formaldehyde (mxaF, xoxF1, and xoxF2) as well as in carbon assimilation (fae1, fae2, metF, and sga) decreased when this strain was grown on methanol vs. methane, indicating that methanotrophs manipulate gene expression in response to growth substrate as well as the availability of copper. Interestingly, growth of M. trichosporium OB3b on methane vs. methanol was similar despite such large changes in gene expression. Finally, methanol-grown cultures of M. trichosporium OB3b also exhibited the "copper-switch." That is, expression of pmoA increased and mmoX decreased in the presence of copper, indicating that copper still controlled the expression of alternative forms of methane monooxygenase in M. trichosporium OB3b even though methane was not provided. Such findings indicate that methanotrophs can sense and respond to multiple environmental parameters simultaneously.