Specifically targeting adenoviral vectors to particular cell/tissue types can be achieved by genetically modifying the adenovirus fiber protein. Two common strategies are: (1) directly modifying the fiber gene in the adenovirus genome and (2) in trans supply of the modified fiber. The former however, suffers from difficulties in directly manipulating large adenoviral genomic DNA. Although the latter allows easy manipulation of the small fiber gene, our studies show that the in trans supplement of the modified fiber causes incomplete fiber assimilation in the virus. Thus an alternate cloning strategy was devised to facilitate the insertion of cell-targeting sequences into the HI loop of a CAR binding-ablated fiber gene in the Ad5 genomic backbone. Our approach retains the advantage of easily modifying the fiber with the additional benefit of genetic re-insertion into the Ad genomic backbone to ensure complete modified fiber incorporation. Using this strategy, an endothelial cell binding peptide sequence (Asn-Gly-Arg) was introduced into the Ad fiber and showed that the generated Ad vector displayed selective transduction of endothelial cells both in vitro and in vivo compared to the conventional vector. Furthermore, this Ad vector cloning strategy can be adapted to introduce other peptide sequences to target other cell types.