We have developed a novel, reproducible, and facile methodology for the construction of artificial lipid envelopes for adenoviruses (Ad) by self-assembly of lipid molecules around the viral capsid. No alteration of the viral genome or conjugation surface chemistry at the virus capsid was necessary, therefore difficulties in production and purification associated with generating most surface-modified viruses can be eliminated. Different lipid bilayer compositions produced artificially enveloped Ad with physicochemical and biological characteristics determined by the type of lipid used. Physicochemical characteristics such as vector size, degree of aggregation, stability, and surface charge of the artificially enveloped Ad were correlated to their biological (gene transfer) function. In monolayer cell cultures, binding to the coxsackie and adenovirus receptor (CAR) was blocked using a zwitterionic envelope, whereas enhanced binding to the cell membrane was achieved using a cationic envelope. Envelopment of Ad by both zwitterionic and cationic lipid bilayers led to almost complete ablation of gene expression in cell monolayers, due to blockage of virion endosomal escape. Alternatively, artificial Ad envelopes built from lipid Mayers at the fluid phase in physiological conditions led to enhanced penetration of the vectors inside a three-dimensional tumor spheroid cell culture model and delayed gene expression in the tumor spheroid compared to nonenveloped adenovirus. These results indicate that construction of artificial envelopes for nonenveloped viruses by lipid bilayer wrapping of the viral capsids constitutes a general strategy to rationally engineer viruses at the nanoscale with control over their biological properties. © 2008 American Chemical Society.