Stable carbon isotope ratios are an important tool for understanding methanogenesis in the environment. When applied to biological methanogenesis, interpretation of carbon isotope ratios requires a thorough understanding of how the availability of different substrates affects the eventual δ 13C of methane, biomass and lipids. Methanosarcina barkeri was grown on four substrates: methanol, trimethylamine (TMA), acetate, and H 2/CO 2, under variable conditions in which the substrate was either present in excess or limited in availability. The extent of isotopic fractionation between the carbon substrate and the products of M. barkeri was dependent on the substrate type and availability. Growth on unlimited substrate resulted in a range of observed isotope fractionation, with growth on methanol yielding methane, biomass and lipids most depleted in 13C relative to substrate, and growth on acetate yielding the least depleted products. Autotrophic fractionations were intermediate. Substrate-limited growth afforded smaller depletions in 13C on all substrates. There were large differences in the δ 13C among the M. barkeri lipids produced within each experiment, with the notable exception of growth on acetate. The 13C content of lipids was generally well correlated with that of biomass, with archaeol showing the strongest relationship. The 13C content of individual lipids varied with substrate availability in some cases, but did not show patterns that could be used to identify the growth substrate of methanogens in natural environments.