Recombinant adenoviruses are efficient gene delivery vectors that are being evaluated in many gene therapy and vaccine applications. Methods for rapid production of ca. 10(12)-10(13) Virus particles (VPs) are desired to enable rapid initial evaluation of such vectors. For this purpose, a scalable transfection procedure was developed for production of an adenovirus type 5 vector expressing HIV-1 gag gene (MRKAd5gag). Adherent PER.C6 cells were transfected by calcium phosphate coprecipitation of the linearized, 36 kb adenovirus plasmid in disposable culture vessels. Various process variables including precipitate formation time, DNA concentration, and harvest time were investigated to rapidly achieve desired virus yields using an adenovirus plasmid encoding the green fluorescent protein (pAd5gfp). Using an optimized procedure, consistent production of >5 x 10(10) VPs per 1-tray Nunc cell factory (NCF) with a ratio of infectious units to virus particles of >1:10 was obtained for the MRKAd5gag vector. This scaleable process can be used to produce adenoviral vectors using several 1-tray NCFs or a single multiple-tray NCF within I month from the time of plasmid construction.