This paper documents the sedimentological setting, mineralogy, and geochemistry of several iron formation units interbedded with siliciclastic strata of the Mesoarchean Witwatersrand Supergroup, well known for its world-class conglomerate-hosted Au-U deposits. Four major iron formation beds, with associated magnetic mudstones, are present in two distinctly different lithostratigraphic associations, namely shale- and diamictiteassociated iron formation. The shale association is represented by the Water Tower and Contorted Bed iron formations in the Parktown Formation of the Hospital Hill Subgroup in the lower part of the succession and the diamictite association by the Promise and Silverfield iron formations in the overlying Government Subgroup. The iron formation units have been subjected to lower greenschist facies metamorphism. Oxide (magnetite and limited hematite), carbonate, and silicate facies iron formations are recognized. The iron formations typically overlie major transgressive flooding surfaces in the succession and, in turn, form the base of progradational coarsening-upward increments of sedimentation comprising magnetic mudstone, nonmagnetic shale, and interbedded siltstone-quartzite. The upward transition from iron formation into magnetic mudstone is accompanied by a change in mineralogical composition from hematite-magnetite iron formation at the base in the most distal setting through magnetite-siderite- and siderite-facies iron formation in the transition zone to magnetic mudstone. The siderite with associated ankerite displays highly depleted δ13C values, suggesting crystallization via iron respiration in presence of organic carbon. The iron formations display positive postArchean Australian shale-normalized Eu and Y anomalies with depletion in light rare-earth elements relative to heavy rare-earth elements, indicating precipitation from marine water with a high-temperature hydrothermal component. Integration of sedimentological, petrographic, and geochemical results indicates that the shale-associated iron formation was deposited during the peak of transgression, when reduced iron-rich hydrothermal waters entered the Witwatersrand Basin over a limited vertical extent due to neutral buoyancy, with the top of the plume occurring below the photic zone. It is suggested that chemolithoautotrophic iron-oxidizing bacteria, which would have been able to exploit the difference in chemistry between the iron-enriched plume water and ambient ocean water to fuel metabolic activity in the presence of limited free molecular oxygen, were responsible for precipitation of initial ferric iron oxyhydroxides. The vertical facies associations in the iron formations most likely developed in response to the limited vertical extent of the hydrothermal plume, with (from distal to proximal) hematite preserved where the base of the plume was not in contact with the basin floor, magnetite where the plume water was in contact with bottom sediment, iron-rich carbonates where organic carbon input was high, iron-rich alumosilicates where siliciclastic input became significant in more proximal settings, and iron-poor sediment above the top of the plume. Diamictite-associated iron formations in the Witwatersrand are inferred to have been deposited in a fashion similar to the shale-associated iron formations, with the exception that major transgressions and hydrothermal plume invasion were preceded by glacial ice cover. The climate warming and increased volcanic activity required could have been related to increased tectonic activity inferred for the Witwatersrand Supergroup during deposition of the glacially associated iron formations.