Adenoviruses (Ad) deleted in the protease (PS) gene are capable of only one round of replication in non-complementing cells. This feature was exploited to develop a positive selection method for constructing adenoviral recombinants using ectopic expression of the PS gene in the E1 region. Very low levels of PS were sufficient to ensure the rescue of a PS-deleted Ad genome (Ad(Delta)PS), thereby eliminating deleterious effects PS over-expression might exert on cell or virus growth. In addition to the standard co-transfection method, an alternative protocol was developed in which the Ad5-(Delta)PS viral DNA was delivered by infection before subsequent transfection of 293 cells with the transfer vector. Under optimal conditions, at least one recombinant Ad per 10(3) cells was generated with 100% of the plaques being recombinant. Since the infection/transfection protocol is readily scalable, this represents the first method that allows for the easy construction of adenovirus vector (AdV) libraries with high diversities. This approach addresses in a novel way the bottleneck encountered when converting plasmid libraries, constructed in E. coli using a variety of well-established strategies, into corresponding AdV libraries. It maintains high diversity while generating recombinant viruses with 100% efficiency.