The microbial contribution to the formation of bound residues in soils is studied by characterizing the metabolic activity of three microorganisms (Trametes versicolor, Fusarium solani and Ralstonia eutropha) on 14C-2,4-dichlorophenoxyacetic acid (2,4-D) during incubation in synthetic liquid media and soil. A fractionation protocol was applied to quantify the 14C-2,4-D that was incorporated into the biomass among biomolecular-like fractions. Successive fractionation of microbial biomass was implemented to break up and quantify the methanol/dichloromethane fraction (corresponding to the 14C-lipid-like fraction), the trichloroacetic acid fraction (or hydrolysed 14C-polysaccharide-like fraction) and the acid hydrolysable fraction (or the hydrolysed 14C-protein-like fraction). Relevant differences in the 2,4-D degradation and biomass radioactivity distribution among the three microorganisms were found. The 14C-protein-like fraction was the most consistent biomass fraction for reflecting the pesticide use capacity of the microorganisms under liquid and soil conditions. 2,4-D and its metabolite 4-chlorophenol were detected in methanol/dichloromethane and trichloroacetic acid fractions of the biomass of microorganisms exhibiting a low capacity to mineralize 2,4-D, thus proving that the microbial participation in the formation of bound residues while conserving the initial pesticide structure under natural soil conditions may be intimately associated with the lipid- and polysaccharide-like constituents. The fractionation protocol differentiates between 14C that is incorporated into biomass as a biomolecular constituent and the pesticide or its metabolites that accumulate in the biomass and thus correspond to the stricto sensu definition of bound residues. © 2017 Elsevier B.V.