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The determination of compound-specific stable isotope ratios is a promising new tool to assess biodegradation of organic compounds in groundwater. In this study, the occurrence of carbon isotope fractionation during dechlorination of tetrachloroethene (PCE) to ethene was evaluated in a PCE-contaminated aquifer and in a microcosm that was based on aquifer material from the site. In the microcosm, all dechlorination steps were accompanied by carbon isotope fractionation. The largest fractionation occurred during dechlorination of cis-1,2-dichloroethene (cDCE) and vinyl chloride (VC), resulting in a large enrichment of ¹³C in the remaining cDCE and VC. Stable carbon isotope ratios (δ¹³C) of cDCE and VC increased from −25.7 to −1.5‰ and −37.0 to −2.5‰, respectively. The δ¹³C of ethene was initially −60.2‰ and approached the δ¹³C of the added PCE (−27.3‰) as dechlorination came to completion. A …