Our objectives were (i) to develop a molecular method to detect mycelia of Tuber melanosporum (black truffle) in soil and (ii) to test for mycelial distribution around two truffle-bearing Quercus ilex trees in a truffle orchard. Isolation of total DNA from soil was performed, followed by PCR amplification with T. melanosporum-specific primers and restriction analysis. To address the detection sensitivity level, soil samples were inoculated with known amounts of gleba of T. melanosporum. The detection limit was >/=11.4 mug of hyphae g(-1) of soil. Mycelium was detected primarily within the area defined by the truffle burn and within the top 35 cm of the soil in all directions from the trees.