SUMMARYAmino acids produced in vitro by the ectomycorrhizal fungusPisolithus tinctoriuswere analyzed by gas-liquid chromatography. Glutamic acid was the major component (21%) of the amino acids in the total and bound pools during the exponential phase of growth. Alanine was the major component (35%) in the free pool during the acceleration phase and in the early days of the exponential phase (33%), but subsequently decreased rapidly. Arginine, an ornithine-cycle intermediate, was present in substantial amounts (~7) in the total and bound pools during the exponential phase, but decreased in the free pool during the exponential phase. Citrulline and ornithine, also ornithine-cycle intermediates, were present in all fractions. The pattern of free ammonia production suggested urease activity resulting from the reduction of arginine. The pattern of amino-acid synthesis exhibited byP. tinctoriussuggests that alanine as well as glutamic acid may be a route by which amino compounds are formed from inorganic nitrogen. The presence of arginine, citrulline, ornithine, and free ammonia in the free pool may be an indication that the ornithine cycle is operative in this fungus. It is not possible to conclude whether the presence of small or large concentrations of ornithine-cycle intermediates, especially arginine, in ectomycorrhizal fungi is critical for the enhancement of nitrogen uptake in ectomycorrhizae.