Many previous studies on transformation of low molecular weight organic substances (LMWOS) in soil were based on applying 14 C and/or 13 C labeled substances. Nearly all these studies used uniformly labeled substances, i.e. all C atoms in the molecule were labeled. The underlying premise is that LMWOS transformation involves the whole molecule and it is not possible to distinguish between 1) the flux of the molecule as a whole between pools (i.e. microbial biomass, CO 2 , DOM, SOM, etc.) and 2) the splitting of the substance into metabolites and tracing those metabolites within the pools.Based on position-specific 14 C labeling, we introduce a new approach for investigating LMWOS transformation in soil: using Na-acetate labeled with 14 C either in the 1st position (carboxyl group, –COOH) or in the 2nd position (methyl group, –CH 3 ), we evaluated sorption by the soil matrix, decomposition to CO 2 , and microbial uptake as related to both C atoms in the acetate. We showed that sorption of acetate occurred as a whole molecule. After microbial uptake, however, the acetate is split, and C from the –COOH group is converted to CO 2 more completely and faster than C from the –CH 3 group. Correspondingly, C from the –CH 3 group of acetate is mainly incorporated into microbial cells, compared to C from the –COOH group. Thus, the rates of C utilization by microorganisms of C from both positions in the acetate were independently calculated. At concentrations of 10 μmol l −1 , microbial uptake from soil solution was very fast (half-life time about 3 min) for both C atoms. At concentrations <100 μmol l −1 the oxidation to CO 2 was similar for C atoms of both groups (about 55% of added substance). However, at acetate concentrations >100 μmol l −1 , the decomposition to CO 2 for C from –CH 3 decreased more strongly than for C from –COOH.We conclude that the application of position-specifically labeled substances opens new ways to investigate not only the general fluxes, but also transformations of individual C atoms from molecules. This, in turn, allows conclusions to be drawn about the steps of individual transformation processes on the submolecular level and the rates of these processes.