Stable Isotope Probing (SIP) is a powerful tool for analysing the fate of pesticides in soil. Together with FAME (Fatty Acid Methyl Esters), it can help identify biodegradation pathways and recycling into the microbial biomass. The fate of ring-labelled C-13-2,4-dichlorophenoxyacetic acid or 2,4-D (C-2,C-4-D) was determined in soil during a 6-month incubation. The distribution of C-13 among the microbial biomass, the CO2 respired, the water, methanol and dichloromethane soluble fractions, and the residual non-extracted bulk soil was measured. Molecular analyses were carried out on the lipid and the non-extractable fractions. After 8 days, about half of the initial amount of C-2,C-4-D was mineralised; the other half remained in soil as non-extractable residues (NER). C-2,C-4-D continued to be mineralised, suggesting that NER were still bioavailable. Analysis of C-2,C-4-D-enriched FAME contained in the lipid fraction suggested that a succession of microbial populations was involved in 2,4-D biodegradation. This is possibly due to the change of 2,4-D availability. The C-2,C-4-D yield coefficient and degrader diversity evolved during the incubation, providing corroboratory evidence that different physiological groups were active during the incubation. The C-13-labelled microbial community was always less diverse than the total community, even at the end of the incubation, suggesting that the cross-feeding community is also a specific part of the total community. This work shows that molecular analysis of C-13-labelled pesticides is a useful tool for understanding both chemical and biological aspects of their fate in soil.