The carbon isotope ratios preserved in sedimentary rocks can be used to fingerprint ancient metabolisms. Organic carbon in Late Archean samples stands out from that of other intervals with unusually low δ^(13)C values (∼−45 to −60‰). It was hypothesized that these light compositions record ecosystem-wide methane cycling and methanotrophy, either of the aerobic or anaerobic variety. To test this idea, we studied the petrography and carbon and oxygen isotope systematics of well-known and spectacular occurrences of shallow water stromatolites from the 2.72 Ga Tumbiana Formation of Western Australia. We examined the carbonate cements and kerogen produced within the stromatolites, because methanotrophy is expected to leave an isotopic fingerprint in these carbon reservoirs. Mathematical modeling of Archean carbonate chemistry further reveals that methanotrophy should still have a discernible signature preserved in the isotopic record, somewhat diminished from those observed in Phanerozoic sedimentary basins due to higher dissolved inorganic carbon concentrations. These stromatolites contain kerogen with δ^(13)C_(org) values of ∼−50‰. By microsampling different regions and textures within the stromatolites, we determined that the isotopic compositions of the authigenic calcite cements show a low degree of variation and are nearly identical to values estimated for seawater at this time; the lack of low and variable δ^(13)C_(carb) values implies that methanotrophy does not explain the low δ^(13)C_(org) seen in the coeval kerogen. These observations do not support a methanotrophy hypothesis, but instead hint that the Late Archean may constitute an interval wherein autotrophs employed markedly different biochemical processes of energy conservation and carbon fixation.