Three different target tissues (protoplast-derived cells, nodules, and stems) and two unrelated hybrid genotypes of Populus (P. alba x P. grandidentata 'Crandon' and P. nigra 'Betulifolia' x P. trichocarpa) have been stably transformed by electric discharge particle acceleration using a 18.7 kb plasmid containing NOS-NPT, CaMV 35S-GUS, and CaMV 35S-BT. Four transformed plants of one hybrid genotype, NC5339, containing all 3 genes were recovered and analyzed. Two expressed GUS and one was highly resistant to feeding by 2 lepidopteran pests (the forest tent caterpillar, Malacosoma disstria, and the gypsy moth, Lymantria dispar.) Pretreatment of the target tissues, fine-tuning of the bombardment parameters, and the use of a selection technique employing flooding of the target tissues were important for reliable recovery of transformed plants.