Ability of replication-defective adenovirus vectors to achieve efficient gene transfer in most of the mammalian cell types makes them useful vehicles for many gene transfer applications, including their use in assessing gene function. High throughput creation of recombinant adenovirus becomes a critical path to the expanding utility of adenovirus vector technology. Here, we report a process in which recombinant adenovirus vectors are isolated as single molecular clones through a convenient direct cloning and green-white selection procedure, and directly transfected into 293 cells where virus is rescued through an enzymatic reaction mediated by an intron-encoding rare endonuclease I-Sce I. This process of enzymatic rescue of circular molecular clones was at least 10-fold more efficient than that using linearized clones for transfection. This method will facilitate a high throughput creation of vectors as required for screening gene function.