T alpha I (thymosin alpha I), an immune booster, plays an important role in the maturation, differentiation and function of T-cells. It can also activate the production of cytokines in dendritic cells. Tal is one of two thymosin proteins that have potential future clinical applications. In order to express T alpha I protein in plants, we designed and synthesized the T alpha I gene according to the plant codon usage bias and created a novel 4 x T alpha I concatemer (four copies of the T alpha I gene arranged end-to-end in tandem, designated 4 x T alpha I). Subsequently, a plant binary expression vector, PG-pRD 12-4 x T alpha I, was constructed and introduced into tomato via Agrobacterium tumefaciens-mediated transformation. Through selection, 54 regenerated tomato plants resistant to kanamycin were obtained, and four transgenic tomato plants were further confirmed by PCR and Southern blotting. RT-PCR (reverse transcription-PCR) analysis showed that the 4 x T alpha I gene was transcribed specifically in tomato [Solanum lycopersicum (formerly Lycopersicon esculentum)] fruits. ELISA analysis showed that the content of the 4 x Tot I protein reached a maximum of 6.098 mu g/g fresh weight in mature tomato fruit. Western-blot analysis further confirmed the expression of 4 x T alpha I protein in transgenic tomato fruits. The MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide] assay showed the 4 x T alpha I protein derived from transgenic tomatoes exhibited bioactivity that can stimulate the proliferation of mice splenic lymphocytes in vitro, and the specific activity of T alpha I protein from the artificial system was higher than that from the synthetic Escherichia coli system. This study is the first to report successful expression of bioactive T alpha I in plants, and also it will provide the basis for furl ther development of the plant system to produce T alpha I.