The concentration and isotopic composition of monochlorobenzene (MCB) was monitored in the plume of an anaerobic, contaminated aquifer in Bitterfeld, Germany. An enrichment in the carbon isotopic composition of more than 4 delta units was found at the fringes of the plume relative to the center (-26.5 %), suggesting the occurrence of in situ biodegradation of MCB. A similar enrichment was measured in a detailed cross-section of the plume and in depth-specific samples obtained in a multilevel sampling well. The latter samples gave a good correlation of MCB concentrations and respective isotopic composition according to the Rayleigh equation. On the other hand, batch experiments using the aerobic MCB-degrading strains Ralstonia sp. DSM 8910, Acidovorax facilis UFZ B517, Rhodococcus erythropolis UFZ B528, and Pseudomonas veronii UFZ B547 showed that the known aerobic pathway initiated by dioxygenases does not result in a significant isotopic fractionation. Thus, a novel anaerobic pathway resulting in an isotopic fractionation appears to be the predominant process of MCB degradation in this aquifer. The study also clearly demonstrates the usefulness of isotopic fractionation analysis to prove biodegradation directly in the field, even when microcosm studies are not available and a metabolic pathway has not yet been elucidated.