Factors affecting isolation and fusion of protoplasts of three Quercus species were investigated and procedures for isolation, purification and fusion of protoplasts of the three species were also established. Unhardened leaves and rapidly growing callus cultures were good source of viable protoplasts. The optimum composition of enzyme mixture for rapid isolation of protoplasts from leaf mesophyll tissues and calli was Cellulase Onozuka R-10 (20g/ℓ, Macerozyme R-10(10g/ℓ), Pectinase(250 units/ℓ, CaCl₂·2H₂O(14mM), MgSO₄·7H₂O(1.8mM), KNO₃(1.0mM), H₃BO₃(1.0mM), KH₂PO₄(0.2mM), KI(1.0μM), 1,4-dithiothreitol (0.1mM) and mannitol (0.6M). Optimum density of protoplasts for maximum fusion was 2×10^5/㎖ which was the highest protoplast density given in this study. Optimum concentration and duration of PEG 1450 treatment for inducing fusion appeared to be 29%(W/V) final PEG 1450 concentration and 5-10 minutes, respectively.