The Ventersdorp Contact Reef, one of the major gold-bearing conglomerate horizons in the Witwatersrand Basin, occurs as a distinct horizon between the overlying Klipriviersberg Group lavas (2714 Ma) and the underlying Central Rand Group rocks (<2894 to >2714 Ma). The Ventersdorp Contact Reef has been metamorphosed and hydrothermally altered under greenschist facies conditions (290–350 °C and 0.2–0.3 GPa). A study of S, O, and H isotopes was carried out to constrain the sources of hydrothermal fluids and gold mineralization in the Ventersdorp Contact Reef. A narrow range of δ³⁴S values (−1.5‰ to +1.8‰) for authigenic pyrite, pyrrhotite, chalcopyrite, sphalerite, and galena from the Ventersdorp Contact Reef and its hanging-wall and footwall lithologies suggests a reconstitution of detrital sulfides during fluid circulation at peak metamorphic conditions. The δ¹⁸O values of authigenic quartz and calcite range from +8.9‰ to +11.3‰, and the δ¹⁸O and δD values of muscovite separates are between +7.2‰ and +8.4‰ and −62‰ and −31‰ respectively. The estimated δ¹⁸O (+4.8‰ to +6.1‰) and δD (−27‰ to −39‰) values of the fluids in equilibrium with these minerals under the relevant P-T conditions suggest a metamorphic origin for the fluid. It is concluded that the metamorphic fluids involved were probably derived from the Basin itself, and allogenic sulfides in the Ventersdorp Contact Reef were reconstituted during fluid circulation at peak metamorphic conditions. Accordingly, gold appears to have been locally remobilized and possibly derived from pre-existing placer concentrations.