[1-¹³C]-labelled phenanthrene was incubated in a closed bioreactor to study the flux and biotransformation of polycyclic aromatic hydrocarbon (PAH) in contaminated soils on a bulk and molecular level. The degradation of extractable phenanthrene was observed by GC–MS measurements and the mineralisation was monitored by ¹³CO₂ production. The transformation of the ¹³C-label into non-extractable soil-bound residues was determined by carbon isotopic measurements. With these data we were able to calculate a carbon budget of the ¹³C-label. Moreover, the chemical structure of non-extractable bound residues was characterised by applying selective chemical degradation reactions to cleave xenobiotic subunits from the macromolecular organic soil matrix. The obtained low molecular weight products yielded ¹³C-labelled compounds which were identified using IRM (isotope ratio monitoring)–GC–MS and structurally characterised with GC–MS. Most of the ¹³C-labelled products obtained by chemical degradation of non-extractable bound residues are well-known metabolites of phenanthrene. Thus, metabolites of [1-¹³C]phenanthrene formed during biodegradation appear to be reactive components which are subsequently involved in the bound residue formation. Hydrolysable amino acids of the soil residues were significantly labelled with ¹³C as confirmed by IRM–GC–MS measurements. Therefore, phenanthrene-derived carbon was transformed by anabolic microbial processes into typical biologically derived compounds. These substances are likely to be incorporated into humic-like material after cell death.