© 2016, American Society for Microbiology. All Rights Reserved. The objective of this study was to characterize metabolically active, aerobic methanotrophs in an ombrotrophic peatland in the Marcell Experimental Forest, in Minnesota. Methanotrophs were investigated in the field and in laboratory incubations using DNA-stable isotope probing (SIP), expression studies on particulate methane monooxygenase (pmoA) genes, and amplicon sequencing of 16S rRNA genes. Potential rates of oxidation ranged from 14 to 17 μmol of CH4 g dry weight soil-1 day-1. Within DNA-SIP incubations, the relative abundance of methanotrophs increased from 4% in situ to 25 to 36% after 8 to 14 days. Phylogenetic analysis of the 13C-enriched DNA fractions revealed that the active methanotrophs were dominated by the genera Methylocystis (type II; Alphaproteobacteria), Methylomonas, and Methylovulum (both, type I; Gammaproteobacteria). In field samples, a transcript-to-gene ratio of 1 to 2 was observed for pmoA in surface peat layers, which attenuated rapidly with depth, indicating that the highest methane consumption was associated with a depth of 0 to 10 cm. Metagenomes and sequencing of cDNA pmoA amplicons from field samples confirmed that the dominant active methanotrophs were Methylocystis and Methylomonas. Although type II methanotrophs have long been shown to mediate methane consumption in peatlands, our results indicate that members of the genera Methylomonas and Methylovulum (type I) can significantly contribute to aerobic methane oxidation in these ecosystems.