ABSTRACT Methanosarcina species possess three operons ( mtaCB1 , mtaCB2 , and mtaCB3 ) encoding methanol-specific methyltransferase 1 (MT1) isozymes and two genes ( mtaA1 and mtaA2 ) with the potential to encode a methanol-specific methyltransferase 2 (MT2). Previous genetic studies showed that these genes are differentially regulated and encode enzymes with distinct levels of methyltransferase activity. Here, the effects of promoter strength on growth and on the rate of methane production were examined by constructing strains in which the mtaCB promoters were exchanged. When expressed from the strong P mtaC1 or P mtaC2 promoter, each of the MtaC and MtaB proteins supported growth and methane production at wild-type levels. In contrast, all mtaCB operons exhibited poorer growth and lower rates of methane production when P mtaC3 controlled their expression. Thus, previously observed phenotypic differences can be attributed largely to differences in promoter activity. Strains carrying various combinations of mtaC , mtaB , and mtaA expressed from the strong, tetracycline-regulated P mcrB ( tetO1 ) promoter exhibited similar growth characteristics on methanol, showing that all combinations of MtaC, MtaB, and MtaA can form functional MT1/MT2 complexes. However, an in vitro assay of coupled MT1/MT2 activity showed significant variation between the strains. Surprisingly, these variations in activity correlated with differences in protein abundance, despite the fact that all the encoding genes were expressed from the same promoter. Quantitative reverse transcriptase PCR and reporter gene fusion data suggest that the mtaCBA transcripts show different stabilities, which are strongly influenced by the growth substrate.