Helper-dependent adenoviral vectors possess a number of characteristics that make them attractive gene therapy vectors. These vectors are completely devoid of viral coding sequences and are able to mediate high-efficiency transduction in vivo to direct sustain high-level transgene expression with negligible chronic toxicity. This review focuses on advances in helper-dependent adenoviral vector technology, selected examples of in vivo studies of particular interest, and the issue of vector-mediated acute toxicity.