Intact viable protoplasts were isolated from leaf mesophyll of Populus nigra x P. maximowiczii with a mean yield of 10.5×10^6 protoplasts per g fresh weight by the enzyme solution with Cellulase 2.0%, Macerozyme 0.8%, Hemicellulase 1.2%, Driselase 2.0% and Pectolyase 0.05%. The protoplasts were cultured on four types of methods; Hanging drop, Liquid plating, Liquid drop and Sieve contained semi-solid agar plating method. The MS basal medium (minus NH₄NO₃) with 0.5 mg.l^(-1) BAP and 2.0 mg.l^(-1) 2.4-D showed the highest frequency of dividing protoplasts by liquid plating method. The percentage of colony formation among four types of culture methods was revealed the highest frequency by the sieve contained semi-solid agar plating method in same culture medium for cell division. Growing cell colonies were transferred to a 0.5% semi-solid agar medium after 12 weeks of culture and callus formation was obtained. Protoplast-derived calluses produced numerous shoots when transferred to regeneration medium of MS mineral salt supplemented with 1.5 mg.l^(-1) zeatin. The roots were induced only 5% to differentiate from the shoots onto a MS medium lacking growth regulators.