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An isolate of Tricholoma matsutake F275 from a fruiting body collected at Tochigi Prefecture in 2003, was successfully grown on a newly developed "HY medium" at 23. The mycelia from medium were then transferred onto substrate of barley grains, which was ramified in 40-50 days after inoculation. Four to five barley grains, covered with mycelia, were put nearby 30-35 days old seedlings of Pinus densiflora (Japanese Red Pine) grown on water gellan gum in test tubes. The observation of root tissue by differential interference microscope revealed that the mycelia of F275 could grow and penetrate into the intercellular space of lateral roots of P. densiflora seedling. This study reports a simplified in vitro method of ectomycorrhiza formation by T. matsutake on seedlings of P. densiflora grown on water gellan gum.