Addition of easily available organic substances to soil often increases the CO2 efflux from pre-existing soil carbon (C). This phenomenon is often explained in terms of the Nitrogen (N)-Mining Hypothesis. According to this proposed – but never conclusively proven – mechanism, increased C availability induces N limitation in microbes, which then access N by degrading soil organic matter (SOM) – a priming effect. This is supported by some experiments demonstrating reduced CO2 efflux after mineral N addition. However, amino acids cause priming, despite their very low C:N ratios and rapid deamination to mineral N. To explore this contradiction, we applied 14C- and 15N-labelled C and N sources (glucose, alanine and ammonium sulfate) to rigorously test two key predictions of the N-Mining Hypothesis: (i) an amino acid should stimulate much less priming than glucose, and (ii) priming should be similarly suppressed for an amino acid or a stoichiometrically equivalent addition of glucose plus mineral N. Both of these key predictions of the N-Mining Hypothesis failed. Efflux of CO2 from native C was essentially determined by the type and amount of C added, with alanine stimulating more priming than glucose (16–50% cumulative increase relative to control, versus 0–25%, respectively). Higher C additions caused more priming than low additions. Mineral N reduced native-C-derived CO2 efflux when added alone or with organic substrates, but this effect was independent of the organic C additions and did not influence C-induced priming. These results were inconsistent with the hypothesized role of N mining in priming. We conclude that the N-Mining Hypothesis, at least in its current form, is not a universal explanation for observed priming phenomena. Instead, we observed a strong correlation between the rates of priming and the mineralization of the added substrates, especially during the first 8 days. This indicated that priming was best explained by energy-induced synthesis of SOM-degrading exoenzymes, possibly in combination with apparent priming from accelerated turnover of microbial biomass. © 2018 Elsevier Ltd