To study the role of intron in the expression of hFIX, retroviral vectors with intron containing hFIX were constructed. It is fundamental for the intron study whether the intron constructed in retroviral vector can be steadily transferred into target cell. First, we constructed two forward-orientation retroviral vectors: G1NaC-i-IX contains the exogenous intron from IL-2, and G1NaC-i'-IX contains the truncated intron I from hFIX gene, covering the splicing donor and acceptor sequences. RT-PCR result indicated that intron in the forward-orientation retroviral vector was spliced after packaging in PA317. Then, reverse-orientation retroviral vectors G1NaC-i'-IXR and G1NaPAIXi' BAM were constructed, in which the reverse and complimentary sequences of hFIX gene with intron appeared in retroviral RNA. RT-PCR assay combined with ELISA test indicated that intron was retained after packaging and hFIX gene with intron constructed in the reverse-orientation retroviral vector can be transduced intact and expressed hFIX at a high level in vitro.