An efficient and robust system of shoot organogenesis for lemon has been developed as an initial step in the transformation and in vitro mutagenesis protocols. In vitro adventitious organogenesis was studied and histologically characterized using mature nodal explants of two important cultivars of Citrus limon, ‘Verna 51’ and ‘Fino 49’. The effects of several plant growth regulator concentrations, incubation conditions, basal medium, explant type and position along the shoot, and ethylene inhibitors were determined. The presence of 1–3 mg L−1 N 6-benzyladenine, in combination with 1 mg L−1 gibberellic acid, was essential for the development of adventitious buds, with the optimum concentration depending on the cultivar, while organogenesis was lowest when N 6-benzyladenine was used in the medium without gibberellic acid. When the effect of different basal media was studied, the best results for percentage of responsive explants were obtained with Murashige and Skoog medium for both cultivars, while the number of buds per regenerating explant was less than 2 in Woody Plant medium. Attempts were made to identify the type of explant that had the highest regeneration ability using mature shoots of C. limon. When different types of explants (nodal segments with the buds completely removed or internodal segments) were compared, the highest percentage of responsive explants was obtained with nodal segments, while with internodal segments, the regeneration percentage was very poor. Histological analysis showed that nodal segments were more reactive than internodal ones and those structural changes occurred sooner in nodal segments. The existence of a morphogenetic gradient along the shoot was also observed and nodal segments from the apical region exhibited the highest regeneration ability. An initial culture period of 2–4 wk in the dark, depending on the cultivar, clearly improved the regeneration percentage and the number of buds for lemon adult explants. Silver thiosulphate increased the regeneration percentage in adult explants of both cultivars, and the best results were obtained with 20 μM silver thiosulphate.