The stable isotopic composition of biogenic methane fluxes from wetland environments may provide important constraints on their role in the global methane budget. However, the causes of observed spatial and temporal variations in delta (del) 13C values are not well understood. The controls on these del 13C values were investigated through field and laboratory experiments. Isotopic shifts associated with methane production could result from changes in: (1) isotopic compositions of the precursors, (2) kinetic isotope effects associated with methane production, (3) pathways of methane production. The purpose of this study was to use rate and isotopic data from incubation experiments employing natural soils and sediments from two contrasting wetlands in North Carolina and Michigan to determine the causes of observed natural variations. Methodologies used to determine rates and pathways in wetlands were critically assessed. Methane produced in sediments from a tidal freshwater estuary site in North Carolina, exhibited relatively small changes in del 13C values throughout the year, ~3 per thousand (mil), with 13C-enrichment occurring during warmer months (winter = -69.2 per mil; summer = -66.1 per mil). The majority (84 +/- 22%) of this seasonal isotopic variation could be attributed to changes in the del 13C value of the sum CO2 pool and the kinetic isotope effect associated with methane production from CO2. A Michigan peatland also displayed 13C-enrichment in methane produced during the warmer months (+11 per mil). This isotopic shift resulted from significant changes in pathways of methane production. The del 13C value of methane from CO2 reduction (-71.4 +/- 1.8 per mil) was depleted in 13C compared to methane produced from acetate (-29.3 +/- 24.4 per mil). Tracer experiments utilizing 14C-labeled substrates indicated that during January, 94 +/- 20% of the methane was produced by CO2 reduction. However, as the sediments warmed during the spring, rates of aceticlastic methanogenesis approached those of CO2 reduction. At both sites, the del 13C values of methane produced during warmer months were enriched in 13C. Small seasonal variations (~ 3-6 per mil) in the del 13C values of biogenic methane from wetlands can result from changes in the del 13C values of substrates. However, larger variations (10 per mil or more) appear to be a result of significant shifts in the balance of production mechanisms.