A fungus (Rs10) symbiotic with orchids was grown in axenic liquid-still culture on mineral–sugar media. Dextrose was limiting at 1% and 2%, with mycelium dry weights directly proportional to the amount of sugar in the medium, but not at 3%. Sucrose gave similar results and was converted into reducing sugars, in the medium, in excess of utilization. With 3% sugar growth was not much better than with 2%, and daily dry weight values were more variable after the active growth phase. With 1% and 2% dextrose in the media, and 10, 20, and 30 ml/flask, dry weight was not affected by the different aeration conditions. Inocula failed to grow on media with pH 3.0, but growth seemed normal with pH 4.5. Ninhydrin-positive substances were not released in the culture media but were found in ethanol extracts of the mycelium. They were more abundant during the active growth phase and on media with 1% vs. 2% dextrose; with less sugar, high levels were maintained for longer periods. They consisted mainly of glutamic acid, glutamine, and aspartic acid, with some fluctuations in concentration related to growth phase. Metabolism seemed different on media with ammonium + nitrate vs. nitrate alone as nitrogen source. The hyphal wall contained chitin.Protocorms of Dactylorhiza purpurella (an orchid with which Rs10 is symbiotic) were grown on eight nutrient media with one amino acid in each, until only the latter were depleted. The best results were with glutamic acid, ornithine, and arginine, followed by aspartic acid and glycine. Alanine, serine, and lysine were toxic. N-acetyl-D-glucosamine derived from chitin was drip-fed to other cultures. It was ineffective at low and toxic at high concentrations.Implications of the results relating to mycorrhizal associations are briefly discussed.