Many applications for human gene therapy would be facilitated by high levels and long duration of physiologic gene expression. Adenoviral vectors are frequently used for gene transfer because of their high cellular transduction efficiency in vitro and in vivo. Expression of viral proteins and the low capacity for foreign DNA limits the clinical application of first- and second-generation adenoviral vectors. Adenoviral vectors with all viral coding sequences deleted offer the prospect of decreased host immune responses to viral proteins, decreased cellular toxicity of viral proteins and increased capacity to accommodate large regulatory DNA regions. Currently most vectors used in vivo for preclinical and clinical studies express cDNAs under the control of heterologous eukaryotic or viral promoters. Using an adenoviral vector with all viral coding sequences deleted and containing the complete human alpha1-antitrypsin (PI) locus, we observed tissue-specific transcriptional regulation in cell culture and in vivo; intravenous injection in mice resulted in high levels of very stable expression for more than ten months and decreased acute and chronic toxicity. These results indicate significant advantages of regulated gene expression using genomic DNA for gene transfer and of adenoviral gene transfer vectors devoid of all viral coding sequences.