Three elite hybrid aspen, Populus grandidentata × P. canescens, P. tremuloides × P. tremula, and P. tremuloides × P. davidiana, have been transformed with Agrobacterium tumefaciens strains LBA4404 and EHA105 carrying kanamycin resistance and GUS genes. The leaves of micropropagated shoots were co-cultivated with Agrobacterium for 65 to 72 hr and then transferred to callus-induction medium with 80–120 mg/L kanamycin in the dark. After 2 weeks, the leaves were transferred to shoot-induction medium under 18-hr photoperiod. Regenerated shoots were verified for transformation by histochemical staining and PCR. Transformed shoots rooted and were transplanted to soil. The three hybrid clones differed widely in their medium requirements for regeneration and in their competence for transformation. The leaves of P. grandidentata × P. canescens callused vigorously on a wide variety of media. In a typical transformation experiment, 30% to 60% of infected leaves produced putatively transformed calli (up to 10 calli per leaf). The origin of these calli and the frequency of shoot formation depended on the Agrobacterium strains. The calli from EHA105-infected leaves produced shoots within six weeks of co-cultivation and at high frequencies (70% to 90%). However, the calli from LBA4404-infected leaves produced shoots more slowly and at much lower frequencies (5% to 10%). Delaying selection for 2 weeks was found to lower the transformation frequency. Putatively transformed calli were obtained from P. tremuloides × P. tremula, and P. tremuloides × P. davidiana hybrids at frequencies of only 2% to 3%. The calli regenerated from P. tremuloides × P. davidiana leaves were very small, but they continued to grow upon being transferred to shoot-induction media and have started to produce shoots. The calli from leaves of P. tremuloides × P. tremula were much larger and they produced shoots more quickly. This transformation protocol is currently being used to introduce rooting genes into these hybrids to improve their rooting from hardwood cuttings.