Expiants for establishing callus cultures originated from in vitro cultured hybrid poplar (Populus alba L. X P. grandidentata Michx.). Plant regeneration was achieved from established callus cultures derived from stem internodes (SI), leaf discs (LD), and root segments (RS). Shoot regeneration from callus occurred within 4 weeks of culture on most of the media tested. Frequency of shoot formation was greatly increased by subculturing the selected organogenic calli on regeneration media. The highest rate of multiple shoot formation (an average number of 7/SI, 11/LD, and 8/RS) was obtained by using 0.05 μM IBA in combination with 22.5 μM 2iP, 22.5 μM zeatin, and 12.5 μM 2iP, respectively. Regenerated shoots were easily rooted in polyterra(™) peat plugs in transparent plastic boxes. The rooted plantlets were subsequently transferred to pots containing an artificial potting mix.