The introduction of nucleic acid sequences into the cells of living animals is the basis of generating transgenic and chimeric animals, which themselves serve as basic tools in genetics and as a foundation for emerging gene therapies, gene vaccines, and antisense oligonucleotide-based therapeutics. The detection of gene-modifying events now requires that large numbers of cells be altered, selected, grown, and analyzed in a time-consuming and labor-intensive sequence of steps. A method for rapid, in situ assessment of the uptake and expression of nucleic acids would thus facilitate evaluation of gene delivery systems and DNA-based therapies. We report on development of a real-time bioluminescent reporter that noninvasively indicates the level of promoter activity in living animals. Such a system differs from existing green-fluorescent-protein-(GFP)-based systems, as our reporters spontaneously emit light without a need for outside light sources. A gene fusion product consisting of HIV-1 LTR (promoter) and firefly luciferase gene complex (reporter) was studied in a transgenic mouse. Photons from thein vivo luciferase reaction were detected by a CCD camera, after transmission through the animal's tissues, and used as an indication of the level of gene expression. This allowed us to estimate, in real time, the extent of promoter activity. We were able to assess activity, both on the surface of the animal as well as in deep tissues. In the skin of living transgenic mice, we assessed the levels of induction of HIV-LTR in response to various stimuli (in murine and human tissues, the HIV-1 LTR promoter can be activated by chemical, photo, and thermal signals). Reduction of photon emission from luciferase expressed in the skin was observed during ATP depletion, suggesting use of bioluminescence as an in situ ATP sensor. Photons emitted from internal organs were also detected externally, indicating that a wide variety of promoters expressed at various tissues sites may be studied using this approach. Use of such a reporter system may facilitate an in vivo assessment of new therapies targeting regulation of viral and host gene expression. Our approach has advantages over dye-producing methods (e.g., GFP), as a low background signal makes near single-event detection possible, the signal is real-time rather than integrated, and no cytotoxic photosensitizing dyes are produced. These studies open a window through which biological processes can be viewed in vivo, illuminating the temporal and spatial distribution of gene expression in animals and humans. Supported by NIH N43-NS-4-2315 & RR-00081, Universitywide AIDS Prog., Packard Foundation (Stanford), and ONR N-00014-94-1-1024.