Mineralisation rates provide valuable information concerning the overall cycling of soil organic N; however, detailed information regarding the pathways preceding the mineralisation of organic substrates remains elusive. We have adopted a molecular approach to open the ‘black box’ of organic N cycling in soil. Stable isotope probing employing compound-specific isotopic analysis was used to trace the fate of N and C within metabolites central to organic N cycling. In time course experiments, 15N and 13C from two dual-labelled amino acid (AA) substrates (U-13C,15N-glutamate and U-13C,15N-glycine) were followed into AAs biosynthesised de novo. In the majority of cases, highly significant differences (P <0.01) were revealed in the magnitude and rate of N and C transfer from the AA substrates to products of central metabolic pathways prior to their loss from the AA pool. By applying linear and non-linear regressions, several important parameters were derived, namely rate constants, magnitude of fluxes and measures of biosynthetic proximity, which describe the rate and magnitude of N and C flux through primary metabolic processes. The significant differences in N and C processing demonstrate a decoupling of the N and C cycles at the molecular level, i.e. after 32 days the magnitude of N flux into newly biosynthesised AAs was twofold greater than that of C from both substrates. We anticipate that the parameters derived will have potential for use in developing detailed models of soil organic N and C processing, the construction of which is founded on the connectivity of the processes fundamental to life.