Whereas it is recognized that most trees have the capacity to take up free amino acids, it is unclear how important amino acid uptake is in situ. In-situ root uptake of isotopically-labeled ammonium, glycine and glutamine were compared between low-nitrogen (N)-availability and high-N-availability temperate forests. Comparable immediate 13C- and 15N-enrichment of fine roots indicated that short-term uptake of amino-acids by tree roots was similar between the two sites. Over the remainder of the experiment, recovery of amino-acid 15N in fine roots was greater, and recovery of 13C lower, in the high-N-availability site. Together with greater rates of conversion of amino-acid-derived 15N into inorganic forms, this indicates that a greater proportion of amino-acid-derived N was taken up after it was converted to inorganic forms by soil microorganisms in the high N availability site. Results from this study suggest that capacity for amino acid uptake by tree roots did not differ between high- and low-N availability sites, but differences in microbial mineralization drove strong differences in the availability of inorganic forms of N between sites. •Amino acids were added to soils of temperate forests of contrasting N availability.•Plant roots at both sites were able to take up intact amino acids.•Mineralization of added amino-acid N was greater at the high N availability site.