Considering the constant improvement of Eucalyptus cloning and the search for new technologies to produce plantlets of this species, somatic embryogenesis has attracted interest from research groups and forestry companies that use advanced genetic breeding and cloning programs. The objective of the present study was to verify the effect of concentrations and sources of calcium, concentrations and effect time of cytokinin BAP and polyamine putrescine on the induction and development of somatic embryos in juvenile explants of Eucalyptus grandis x E. urophylla. Cotyledonary explants were inoculated into culture medium containing calcium chloride (MS medium) or calcium nitrate (JADS medium) as source of calcium. Different concentrations of calcium were also used, for MS medium containing: 4.40 gL-1 (control-Ca), 6.60 gL-1 (50% increase over control - Ca 50) and 8.80 gL-1 (increase of 100% over control – Ca 100) of calcium nitrate; and for JADS medium containing: 11.81 gL-1 (Ca), 17.72 gL-1 (Ca50) and 23.62 gL-1 (Ca100) of calcium chloride. Cotyledon explants were inoculated into the primary induction medium (PIM) containing 20.71 μM picloram as growth regulator. At 10, 20 and 30 days of primary induction, the explants were transferred to the secondary induction medium (SIM) containing 20.71 μM picloram and 11.10 μM BAP or 28.36 μM putrescine. The culture medium containing calcium nitrate provided higher callogenesis when compared to the medium containing calcium chloride. The increase in calcium concentration in the media did not provide higher percentage of induction of somatic pro-embryos. However, the addition of 28.36 μM putrescine to the culture medium provided a higher percentage of induction of somatic embryogenesis. The number of somatic pro-embryos formed per explant was higher when BAP and putrescine were added to the culture medium when compared to medium containing only picloram. To obtain a greater number of somatic pro-embryos of Eucalyptus grandis × E. urophylla, the JADS culture medium containing 28.36 μM putrecine should be used.