Dissolved organic nitrogen (DON) and in particular free amino acids represent a key pool in the terrestrial soil C and N cycle. The factors controlling the rate of turnover of this pool in soil, however, remain poorly understood. We investigated the factors regulating the rate of amino acid turnover at different depths (up to 1.2 m) in five low-input, acid soil profiles. Within the root zone (0–60 cm), amino acids constituted 8% of the DON and represented only a small fraction of plant available N. In all the soil profiles, the rate of amino acid mineralisation decreased progressively with depth. The average half-life of the exogenously added amino acids in the soil was 5.8 h in topsoils (0–10 cm), falling to 20 h at a depth of 50 cm and to 33 h at 100 cm. Generally, the rate of amino acid mineralisation correlated positively with total soil C and N, soil microbial activity (basal soil respiration rate) and soil NO3-NO3− content. The relatively rapid rates of microbial amino acid assimilation in subsoils below the root zone (>60 cm) indicate that long-term transport of amino acids (e.g. from soil to freshwaters) will be low. Based upon the C-to-N ratio of the amino acid substrate and the microbial C assimilation efficiency, we estimate that approximately 40–60% of the amino acid-N will be excreted as NH4+NH4+. In conclusion, the rapid rate of free amino acid turnover and their low concentration in soil solution indicate that the formation of inorganic N (NH4+NH4+ and NO3-NO3−) in soil is limited primarily by the rate of free amino acid appearance in soil and not by the rate of amino acid mineralisation.