We have demonstrated expression of bacterial genes transferred into cells of Populus nigra X P. maximowczii by Agrobacterium tumefaciens strains A281 (PGA 472). We determined the optimum concentration of kanamycin sulfate for effective selection of punctured leaf transformed using Agrobacterium binary vector PGA 472 containing a neomycin phosphotransferase gene (NPT-II) which confers kanamycin resistance. A relatively low concentration (10 ㎎/l) of kanamycin sulfate inhibited callus induction from punctured leaf. The combination of cefatoxime (200 ㎎/l) and carbenicillin (300 ㎎/l) showed good performance of discarding Agrobacterium from inoculated punctured leaf. The callus derived from punctured leaf were not established on MS basal medium supplemented with over 10 ㎎/l kanamycin sulfate, while the growth of callus was severely inhibited with 30 ㎖/l kanamycin sulfate. Transformed kanamycin resistant calli were obtained by culturing Agrobacterium inoculated punctured leaf on selective regeneration medium. The transformed kanamycin-resistant callus continued to grow on regeneration media supplemented with kanamycin sulfate to levels of 10 to 50 ㎎/l. The growth of not co-cultured calli was severely inhibited on regeneration medium containing 30 ㎎/1 kanamycin sulfate. Number of shoots regenerated from co-cultured punctured leaf was 3.0, while that of not co-cultured punctured leaf was none. The rate of gene transformation showed 1.7% from the number of shoots regeneration of co-culturing callus. Regenerated shoots are developing from micropropagation for southern blot analysis and inhertance of the kanamycin resistance trait (NPT-II) in the progeny regenerated shoots.