Rising atmospheric carbon dioxide (CO2) is expected to increase forest productivity, resulting in greater carbon (C) storage in forest ecosystems. Because elevated atmospheric CO2 does not increase nitrogen (N) use efficiency in many forest tree species, additional N inputs will be required to sustain increased net primary productivity (NPP) under elevated atmospheric CO2. We investigated the importance of free amino acids (AAs) as a source for forest N uptake at the Duke Forest Free Air CO2 Enrichment (FACE) site, comparing its importance with that of better-studied inorganic N sources. Potential proteolytic enzyme activity was monitored seasonally, and individual AA concentrations were measured in organic horizon extracts. Potential free AA production in soils ranged from 190 to 690 nmol N g−1 h−1 and was greater than potential rates of soil NH4+ production. Because of this high potential rate of organic N production, we determined (1) whether intact AA uptake occurs by Pinus taeda L., the dominant tree species at the FACE site, (2) if the rate of cycling of AAs is comparable with that of ammonium (NH4+), and (3) if atmospheric CO2 concentration alters the aforementioned N cycling processes. A field experiment using universally labeled ammonium (15NH4+) and alanine (13C3H715NO2) demonstrated that 15N is more readily taken up by plants and heterotrophic microorganisms as NH4+. Pine roots and microbes take up on average 2.4 and two times as much NH4+ 15N compared with alanine 15N 1 week after tracer application. N cycling through soil pools was similar for alanine and NH4+, with the greatest 15N tracer recovery in soil organic matter, followed by microbial biomass, dissolved organic N, extractable NH4+, and fine roots. Stoichiometric analyses of 13C and 15N uptake demonstrated that both plants and soil microorganisms take up alanine directly, with a 13C : 15N ratio of 3.3 : 1 in fine roots and 1.5 : 1 in microbial biomass. Our results suggest that intact AA (alanine) uptake contributes substantially to plant N uptake in loblolly pine forests. However, we found no evidence supporting increased recovery of free AAs in fine roots under elevated CO2, suggesting plants will need to acquire additional N via other mechanisms, such as increased root exploration or increased N use efficiency.