Stable isotope measurements are a powerful tool in soil and ecological research. Natural abundance measurements of soil N pools have the potential to provide information regarding N-transformations. Recently we have developed a protocol to measure the ¹⁵N natural abundance of the soil microbial biomass using the chloroform fumigation-extraction procedure. We measured the ¹⁵N isotope signatures of the microbial biomass, extractable and total N pools in soils from an elevation gradient, a semi-arid and tropical soil age gradient, and a dung gradient. Results show that 1) the δ¹⁵N of the microbial biomass differs from that of the soil extractable and total N, and 2) the difference between the natural abundance ¹⁵N composition of the microbial biomass relative to that of the extractable N pool varies with C and N availability in the soil. We postulate that the ¹⁵N-enrichment of the microbial biomass relative to other soil pools is associated with fractionation during N assimilation and dissimilation processes. This interpretation suggests that a high ¹⁵N enrichment of the microbial biomass is associated with low C availability, high dissimilation and thus high N-mineralization rates.