The cultivation and competitiveness of Tuber borchii was investigated. Seedlings of Pinus pinea were infected either with T. borchii, Laccaria bicolor (strain S238), Hebeloma sinapizans, T. borchii and L. bicolor, or T. borchii and H. sinapizans. Uninoculated seedlings weakly infected with Sphaerosporella brunnea were used as controls. In 1990 the seedlings were transplanted to a coastal site at Marina di Ravenna, Italy. Examination of seedling roots in 1994 and 1995 showed that L. bicolor, H. sinapizans and S. brunnea ectomycorrhizas were replaced by T. borchii and other ectomycorrhizal fungi. The DNA sequence of the ITS region was analysed using the specific pair of primers, ITS1–ITS4, to type the ectomycorrhizas of Tuber borchii. The ectomycorrhizas were also identified by digestion of the PCR products using different restriction enzymes. T. borchii truffle production began in 1994 and for the next four years an average of 7 kg of truffles ha−1 were harvested. This study describes a simple method for the cultivation of T. borchii.