Eucalyptus grandis is the most widely used species in planted forests in tropical and subtropical areas. The traits of interest underlying Eucalyptus breeding programs concern productivity and wood quality for the pulp and paper industry, as well as biotic and abiotic stress tolerance. The development of an efficient transformation protocol is necessary to explore eucalypt resources through functional genomics and biotechnology. However, to achieve this goal, an efficient regeneration protocol is still needed. The main purpose of this work was to develop an efficient regenerationtransformation method for Eucalyptus grandis. To this end, we first concentrated our efforts on establishing an ideal micropropagation protocol to obtain good quality explants. Thirty in vitro culture conditions were tested, including different plant growth supplementations and media. The best result came from MS medium supplemented with 0.5 mg/l Zea and 0.2 mg/lNAA. The second step was to develop a reproducible regeneration system through organogenesis in E. grandis. To accomplish this goal, the following conditions were tested: a) different range of cytokinins (Zea, BAP, TDZ, Kin) and auxins (2,4D, NAA, IBA) in 50 different plant growth regulator combinations; b) different types of explants, obtained from micropropagated shoots (e.g. stem, leaf, or root segment) or derived from seedlings (e.g. hypocotyles with or without shoot apex, cotyledons, or roots); c) the effects of explant orientation; and d) different basal media (MS, WPM, ½ WPM). The highest rate of shoot regeneration (38%) occurred when using internode explants incubated on PIMMS followed by 2 mg/l BAP plus 1.0 mg/l NAA. After one month, stem explants were transferred to SIMMS with 4.0 mg/l BAP plus 0.2 mg/l NAA. Using leaf explants, a shoot regeneration rate of 20% was achieved when explants were cultured in 1.0 mg/l Zea plus 0.1 mg/l NAA in MS medium. All the regenerated shoots were rooted into MS medium (half strength) supplemented with 2 mg/l IBA. An important output of this study was the confirmation of the influence of seasons on the shoot regeneration ability of E. grandis. Finally, the transformation of E. grandis from clonal and seedling explants was done using the EHA105 Agrobacterium strain harboring the pMP2482 plasmid. This plasmid has a construct containing the coding sequences for the green fluorescent protein (GFP) and the -glucoronidase protein (GUS). The transformation frequency was 3.75% when root organogenesis was achieved. Transgenesis was verified by GFP visualization and histochemical detection of GUS activity. However, during the time course of this work, non transgenic shoots were recovered. In conclusion, the present study provides an efficient micropropagation and regeneration protocol designed to support the development of a reliable transformation method for E. grandis.