The in vitro performance of herbaceous and woody nodal segments from adult plants and the effect of hydrolyzed casein (HC 500 mg L-1), kinetin (KIN; 6-furfurylaminopurine 0.46 and 4.65 µM) and activated charcoal (AC 1.5 g L-1) were evaluated upon new shoots induction and development, and to establish a system of in vitro propagation from adult plants of Ginkgo biloba. Woody nodal segments did not produce axillary shoots and presented 100% of bacterial and fungal contamination in culture. However, nodal segments from herbaceous shoots were successfully disinfected and displayed high in vitro morphogenic capacity. The HC was essential for the axillary shoots induction and further multiplication, stimulating shoot formation in 85% of the cultured nodal segments and multiple shoots induction in 35% of them at establishment stage. During the multiplication stage, 66.6% of propagules formed new shoots and 33.3% of them formed multiple shoots when cultured with HC. The KIN and AC inhibited the organogenic process in ginkgo. Two distinct patterns of sprouts development were observed in vitro, similar to what occurs in vivo: 1) short shoots with crowded internodes and expansion of only a few leaves and slow growth; 2) long shoots with separated nodes and marked apical growth. This is the first report of multiple shoots in vitro formation in nodal segments obtained from adult plants of Ginkgo biloba.