The leaf mesophyll protoplasts of Populass alba × glandrdosa were isolated from leaf of plantlet in vitro and cultured for plant regeneration. The MS medium (minus NH₄NO₃) with 0.5 ㎎/ℓ BAP and 2.0 ㎎/ℓ 2, 4-D showed the moderate frequency of dividing protoplasts cultured by the lipuid plating method during the first week of culture. The percentage of colony formation was revealed the highest frequency by the gauze contained semi-solid agar plating method after 5 weeks cultured. Ridding out the gauze, the micro-callus was formed on the same semi-solid medium in 8 weeks after protoplasts culture. For proliferation of callus, mini-callus was transferred on the MS solid medium with 0.5 ㎎/ℓ 2, 4-D and 0.1 ㎎/ℓ BAP 12 weeks after culture. Shoot regeneration occurred when the calli derived from protoplasts were cultured on MS medium with 1.0 ㎎/ℓ zeatin and such shoots could be readily rooted on the one half strengthen MS medium with non-phytohormone. Rooting shoots were planted in green-house 22 weeks after protoplast culture.