Thin carbonaceous laminations preserved in shallow-water facies of the 3416 Ma Buck Reef Chert, South Africa, have been interpreted to represent some of the oldest-known mats constructed by photosynthetic microbes. Preservation of these mats within a unit containing facies deposited at water depths ranging from 0 m to >200 m provides an opportunity to explore the electron donors employed in early microbial photosynthesis. The presence of siderite (FeCO3) as a primary sediment, lack of hematite (Fe2O3), and lack of cerium anomalies throughout the Buck Reef Chert imply that the entire water column was anoxic despite the presence of photosynthetic organisms. Authigenic uranium (Ua = U–Th/3) correlates inversely with siderite abundance, suggesting that variations in carbonate rather than oxygen activity controlled uranium mobility. The inferred lack of oxygen and ferric minerals and the presence of dissolved Fe2+ in the water column imply that H2O, Fe2+, and H2S could not have served as primary electron donors for carbon fixation. It is most likely that Buck Reef Chert bacteria utilized H2 as the primary reductant for photosynthesis.