Efficient transformation of lilies is required for their genetic improvement in ornamental and marketable qualities. Although Lilium longiflorum can be transformed by particle bombardment and Agrobacterium, the transformation frequency is low. In this study, we tested new Agrobacterium-mediated transformation methods using shoot segments combined with two different regeneration systems. Shoot segments were co-cultivated for 2 d with Agrobacterium tumefaciens strain AGL1/pCAS04 harboring a binary vector carrying the neomycin phosphotransferase II driven by a promoter from the maize ubiquitin gene. The effect of different concentrations of 6-benzyladenine (BA) and 2,4-dichlorophenoxyacetic acid (2,4-D) on regeneration was investigated. The results indicated that Murashige and Skoog (MS) medium with 4.4 mu M BA and 0.5 mu M alpha-naphthalene acetic acid was optimal for shoot formation, and the nodal stem was the best explant for shoot induction. MS medium with 9.0 mu M 2,4-D and 0.4 mu M BA was optimal for callus induction. The direct shoot formation method regenerated 187 plantlets per 100 explants, and 74.4% of the regenerants were positive in transgene PCR. The callus regeneration method regenerated 20 plantlets per 100 explants, and 31.5% of them were PCR positive. Southern blotting confirmed the insertion of transgene in the plant host genome. The direct shoot formation method is more than 20-fold more efficient than previously reported transformation method in this species.