We recently showed that the ¹⁵N enrichment of the microbial biomass, relative to the soil soluble fraction, correlates negatively with C and N availability and positively with net N mineralization. However, C and N availability and N mineralization rates have a high temporal variability. We investigated how C and N concentrations of the soil total, soluble and microbial pools and their respective δ¹⁵N isotope signature changed over time using a semiarid substrate age gradient in northern Arizona. We observed that although these variables changed significantly over time, the relationship between the ¹⁵N enrichment of the microbial biomass and the C and N availability was maintained. These results suggest that ¹⁵N enrichment of the microbial biomass may be a useful index of soil net N mineralization over time scales relevant to plant growth.