Recombinant adenoviral vectors have been generated either by the in vivo homologous recombination method or by the in vitro direct ligation method. However, the efficiency of adenoviral vector construction by these methods is low, because of the large size of the recombinant vectors. To improve the ease of constructing adenoviral vectors, we used the circular form of adenoviral DNA, which can generate infectious viruses with an efficiency comparable to that of virion DNA, after transfection into 293 cells constitutively producing adenovirus E1 protein. We replaced the E1 region of the circular form of adenoviral DNA with a cosmid vector flanked by loxP sites, resulting in a 41-kb cosmid, designated pALC. An expression cassette that bicistronically expresses IL-5 and green fluorescent protein (GFP) was readily inserted between the loxP-flanked cosmid backbone and the adenoviral genome of pALC, using the cosmid vector cloning system. Transfection of the resulting cosmid into 293 cells did not produce any infectious adenoviruses because its size (46 kb) was larger than the packing capacity of the adenoviral particles. However, cotransfection of a Cre-expression plasmid with this cosmid into 293 cells efficiently excised the loxP-flanked cosmid vector backbone, and produced the adenoviral vector expressing IL-5 and GFP. To simplify our method further, we have produced a 293 cell line constitutively expressing Cre recombinase. Transfection of pALC cosmid alone into this cell line efficiently generated adenoviral vector. The adenoviral vector construction method presented here is simple and efficient and should further facilitate the application of recombinant adenoviral vectors for in vivo and in vitro gene transfer.