Abstract: Objective To efficiently construct a replication-defective recombinant adenoviral vector using a two-step transforma-tion procedure. Methods Plasmid pAdEasy-1 was linearized and transformed into E.coli BJ5183 to construct BJ5183pAdEasy-1 cells. Cytosine deaminase (CD) gene was obtained from plasmid pBS-CD, and subcloned into the shuttle plasmid to form transfer plamid of pAdtrackCMV-CD, which was then linearized and transformed into BJ5183pAdEasy-1 cells. The recom-binant adenovirus plasmid DNA was extracted from the transformed bacteria and digested with PacⅠ after identification, followed by transfection into 293 packaging cells. PCR was used to detect target gene, and the titer and the infection rate of the recombinant Ad were measured with the aid of green fluorescent protein (GFP) reporter gene. The same recombinant AdCMV-CD was constructed in a one-step transformation method for comparison. Results The homologous recombination of the two-step transformation method resulted in a success rate of 76.5% (13/17), while the success rate of the one-step method was only 11.8% (2/17), showing significant difference between the two methods (P=0.000 17 by Fisher’s exact test). Conclusion The two-step transformation procedure is more efficient and convenient than one-step method.