Protoplasts isolated from leaf tissue of P. koreana x nigra were fused with mesophyll protoplasts of P. euramericana cv. Guardi. For the isolation of mesophyll protoplasts, well expanded young leaves from five to seven weeks old seedlings grown in vitro were used as experimental materials. Before enzymatic digestion, leaf tissues were preconditioned at dark room for a day. Both P. koreana x nigra and P. euramericana cv. Guardi were digested in enzyme solution mixed 2.0% Cellulase, 0.8% Macerozyme, 1.2% Driselase and 0.05% Pectolyase. For the purpose of protoplast purification, crude protoplasts were floated on 0.6M sucrose solution containing CPW inorganic salts by centrifuge. Protoplasts were mixed at 1:1 ratio with a density of ×10^6 /㎖. To optimize the condition for the increase of fusion frequency, concentrations and incubation time of fusogen, and pH were examined. PEG and Dextran (MW: 40,000) used as a fusogen were tested in range of 20, - 50% and 15 - 25%, respectively. When protoplasts were treated at the PEG 40% or Dextran 15% for 20 min, the fusion frequency was high by 21%. In addition, Fusion frequency was enhanced at the fusion solution containing 30 mM CaCl₂.2H₂O and adjusted at pH 10.5. Fusion-treated protoplasts were cultured in KM8p liquid medium containing 0.6 M sucrose, 2,4-D 1.0 ㎎/ℓ, and BA 0.1 ㎎/ℓ. The cell division and colony formation were good when protoplasts were cultured by stationary condition and the formation of micro-calli grew well when cell colony was suspended by low speed.