Free amino acids (FAAs) in soil are an important N source for plants, and abundances are predicted to shift under altered atmospheric conditions such as elevated CO2. Composition, plant uptake capacity, and plant and microbial use of FAAs relative to inorganic N forms were investigated in a temperate semiarid grassland exposed to experimental warming and free-air CO2 enrichment. FAA uptake by two dominant grassland plants, Bouteloua gracilis and Artemesia frigida, was determined in hydroponic culture. B. gracilis and microbial N preferences were then investigated in experimental field plots using isotopically labeled FAA and inorganic N sources. Alanine and phenylalanine concentrations were the highest in the field, and B. gracilis and A. frigida rapidly consumed these FAAs in hydroponic experiments. However, B. gracilis assimilated little isotopically labeled alanine, ammonium and nitrate in the field. Rather, soil microbes immobilized the majority of all three N forms. Elevated CO2 and warming did not affect plant or microbial uptake. FAAs are not direct sources of N for B. gracilis, and soil microbes outcompete this grass for organic and inorganic N when N is at peak demand within temperate semiarid grasslands.