Molecular imaging has its root in nuclear medicine and gene therapy monitoring. Therefore, recent progress in the development of non-invasive imaging technologies, particularly nuclear medicine, should allow molecular imaging to play a major role in the field of gene therapy. These tools have recently been validated in gene therapy models for continuous quantitative monitoring of the location, magnitude, and time-variation of gene delivery and/or expression. This article reviews the use of radionuclide imaging technologies as they have been used in imaging gene delivery and gene expression for gene therapy applications. The studies published to date lend support that noninvasive imaging tools will help to accelerate pre-clinical model validation as well as allow for clinical monitoring of human gene therapy. (Nucl Med Mol Imaging 2006;40(2):96-105)