The anaerobic biodegradation of hydrocarbons at mineral oil contaminated sites has gathered increasing interest as a naturally occurring remediation process. The aim of this study was to investigate biodegradation of hydrocarbons in laboratory aquifer columns in the absence of O 2 and NO 3 y , and to calculate a mass balance of the anaerobic biodegradation processes. The laboratory columns contained aquifer material from a diesel fuel contaminated aquifer. They were operated at 258C for 65 days with artificial groundwater that contained only SO 2y and CO 42 as externally supplied oxidants. After 31 days of column operation, stable concentration profiles were found for most of the measured dissolved species. Within 14 h residence time, about 0.24 mM SO 4 2y were consumed and dissolved Fe II Ž.Žup to 0.012 mM ,.Mn II Ž.Žup to 0.06 mM ,.and CH 4 Žup to 0.38 mM were produced. The alkalinity and the dissolved inorganic carbon .ŽDIC concentration . increased and the DIC became enriched in 13 C. In the column, n-alkanes were selectively removed while branched alkanes persisted, suggesting a biological degradation. Furthermore, based on changes of concentrations of aromatic compounds with similar physical-chemical properties in the effluent, it was concluded that toluene, p-xylene and naphthalene were degraded. A carbon mass balance revealed that 65% of the hydrocarbons removed from the column were recovered as DIC, 20% were recovered as CH 4 , and 15% were eluted from the column. The calculations indicated that hydrocarbon mineralization coupled to SO 4 2y reduction and methanogenesis contributed in equal proportions to the hydrocarbon removal. Hydrocarbon mineralization coupled to ) Corresponding author. Tel.: q41-1-633-6042; fax: q41-1-633-1122; e-mail: hoehener@ito.umnw.ethz.ch 1 Present address: BMG Engineering AG, CH-8952 Schlieren, Switzerland.Published in Journal of Contaminant Hydrology 32, issue 1-2, 1998 which should be used for any reference to this work Fe III Ž.and MnŽIV.reduction was of minor importance. DIC, alkalinity, and stable carbon isotope balances were shown to be a useful tool to verify hydrocarbon mineralization.