The filametous ascomycete Tuber borchii is a plant-symbiotic ectomycorrhizal microrganism with an high value due to the production of hypogeous fruitbodies (truffles). The present work was undertaken to develop a procedure for the release of T. borchii viable protoplasts from Tuber mycelium, isolate ATTC 96540; several factors which affect the isolation, morphology and viability were examined and developed in order to improve applications of T. borchii protoplasts in morphological, biochemical and genetic investigations (protoplast fusion or transformation). Functional delivery of liposome content into T. borchii protoplasts has also been examined with a cytotoxic ribosome inactivator as saporin. T. borchii protoplasts incubation/fusion with saporin containing liposomes were made to demonstrate the absence of cell wall of 16 days cultured protoplasts.