This experiment was conducted to identify the optimum conditions for shoot multiplication and isolation and culture of mesophyll protoplasts from Populus alba × P. glandulosa. The suitable concentration of BAP for shoot multiplication was 0.4 ㎎/ℓ. High yield and viability of isolated protoplasts were obtained by shortperiod enzyme treatment from in vitro grown mesophyll tissue of Populus alba × P. glanduiosa. The optimum enzyme concentration for mesophyll protoplast isolation were Cellulase 2.0%, Macerozyme 0.8%, Hemicellulase 1.2%, Driselase 2.0%, Pectolyase Y-23 0.05%. The most effective for protoplast isolation and viability was 0.6m mannitol in enzyme solution. The optimum pH of enzyme solution was 5.6, and the effect of DTT and MES buffer was significant for protoplast purification, the most favorable sucrose concentration of floating solution was important. In addition to normal protoplasts, megaprotoplasts were also observed. Cell division was observed in. MS liquid medium supplemented with NAA 2.0㎎/ℓ and BAP 0.5㎎/ℓ after 7 days.