Ion microprobe measurements of carbon isotope ratios were made in 30 specimens representing six fossil genera of microorganisms petrified in stromatolitic chert from the ∼850 Ma Bitter Springs Formation, Australia, and the ∼2100 Ma Gunflint Formation, Canada. The δ13CPDB values from individual microfossils of the Bitter Springs Formation ranged from −21.3 ± 1.7‰ to −31.9 ± 1.2‰, and the δ13CPDB values from microfossils of the Gunflint Formation ranged from −32.4 ± 0.7‰ to −45.4 ± 1.2‰. With the exception of two highly 13C-depleted Gunflint microfossils, the results generally yield values consistent with carbon fixation via either the Calvin cycle or the acetyl-CoA pathway. However, the isotopic results are not consistent with the degree of fractionation expected from either the 3-hydroxypropionate cycle or the reductive tricarboxylic acid cycle, suggesting that the microfossils studied did not use either of these pathways for carbon fixation. The morphologies of the microfossils suggest an affinity to the cyanobacteria, and our carbon isotopic data are consistent with this assignment.