In this study, compositions and δ13C and δ2H isotopic values of hydrocarbon gases from 5 mines in the Witwatersrand basin, South Africa, support the widespread occurrence of microbially produced methane in millions of years-old fissure waters. The presence of microbial methane is, to a large extent, controlled by the geologic formations in which the gases are found. Samples from the Witwatersand Supergroup have the largest microbial component based on δ13C and δ2H signatures and CH4/C2+ values. Based on mixing between a microbial CH4 component and a more 13C-enriched and 2H-depleted C2+-rich end member, conservative estimates of the % contribution of microbial CH4 to the gas samples range from >90% microbial CH4 at Beatrix, Masimong, and Merriespruit, to between 5 and 80% microbial CH4 at Evander, and <18% microbial CH4 at Kloof. The Witwatersrand basin s history of thermal alteration of organic-rich ancient sedimentary units suggests a thermogenic origin for this 13C-enriched end member. Alternatively, the potential for an abiogenic origin similar to hydrocarbon gases produced by water-rock interaction at other Precambrian Shield mines is discussed. Microbial methane is predominantly found in paleo-meteoric fissure waters with δ18O and δ2H values that fall on the meteoric waterline, and have temperatures between 30 to 40°C. In contrast, fissure waters with a larger component of nonmicrobial hydrocarbon gases show a trend towards more enriched δ18O and δ2H values that fall well above the meteoric waterline, and temperatures of 45 to 60°C. The enrichment in 18O and 2H in these samples, and their high salinity, are similar to the isotopic and compositional characteristics of saline groundwaters and brines produced by water-rock interaction at Precambrian Shield sites elsewhere. The reported 100 Ma ages of fissure waters from the Witwatersrand and Ventersdorp formations suggest that these microbial hydrocarbon gases are the product of in situ methanogenic communities in the deep subsurface of the Witswaterand basin. Small subunit ribosomal RNA genes were amplified using archaeal-specific primer sets from DNA extracts derived from several of these waters. Fissure waters with a high proportion of microbial methane also contained sequences resembling those of known methanogens. Copyright © 2004 Elsevier Ltd.