The quantitative contribution of bacteria to total microbial uptake of nitrogenous substrates is an aspect of the aquatic nitrogen cycle that is still largely unclear, mainly because existing methods are generally inadequate. We investigated the feasibility of measuring 15N incorporation into bacterial D-amino acids by gas chromatography- combustion-isotope ratio mass spectrometry (GC-c-IRMS) and the potential of this method as a new tool for quantification of 15N uptake by bacteria. The presented method allowed analysis of 15N incorporation into various hydrolysable amino acids (HAAs), including the bacterial biomarker D-alanine (D-Ala), at trace levels. The potential of the method was tested in a 15N labeling experiment in which sediment slurries were incubated with 15NH4+ and a 15N-labeled amino acid mixture. 15N incorporation into D-Ala was used to calculate total bacterial 15N uptake while comparison of 15N incorporation into D-Ala versus L-Ala provided a direct measure for the relative contributions of bacteria versus algae to the total microbial 15N uptake. Subsequently, it was also possible to calculate 15N uptake by algae. Results for the test experiment showed that bacteria accounted for 38% of total 15NH4+ uptake and dominated uptake of the 15N-amino acid mixture (90%). Analysis of 15N incorporation into other (non-biomarker) HAAs yielded useful additional information on the transformation of these HAAs during organic matter degradation. In conclusion, GC-c-IRMS analysis of D-Ala combined with 15N labeling is a unique approach in aquatic sciences that provides a powerful new method for quantification of nitrogen flows through bacteria in natural microbial communities.