Plants and microorganisms intensely compete for nitrogen (N) at many stages of the terrestrial N cycle. In particular, the dissolved organic N (DON) pool, and competition for low molecular weight dissolved organic N (LMWDON) compounds such as amino acids and peptides (and LMW dissolved organic matter; LMWDOM as a whole) has received significant recent research interest. However, as LMWDON compounds contain both N and carbon (C), a question that remains is whether soil microorganisms are primarily taking up LMWDON mainly for the C or the N contained therein. We investigated microbial uptake rates of the model peptide l-trialanine as a rapidly cycling LMWDON compound in temperate grassland soils of differing fertility using 14C labelling to assess how soil fertility status influenced microbial uptake of LMWDON. We then imposed an excess of C as glucose and/or N as NH4Cl to ask whether the uptake of the peptide was affected by C or N excess. Our results demonstrate that l-trialanine is taken up rapidly from the soil solution (t½ <1.5 min), and that an excess of C, rather than N, resulted in a reduced uptake of the peptide. From this, we conclude that LMWDON is taken up primarily to fulfil the C requirement of soil microorganisms, indicating that they exist in a C-limited state, and are able to respond quickly to a transient influx of an easily metabolisable resource.