In vivo bioimaging of transgenic luciferase in the lung and nose is an expedient method by which to continually measure expression of this marker gene after gene transduction. Its substrate, luciferin, is typically injected into the peritoneal cavity before bioimaging. Here we demonstrate that, compared with intraperitoneal injection, intranasal instillation of luciferin confers approximately an order of magnitude increase in luciferase bioluminescence detection in both lung and nose. This effect was observed after administration of viral vectors based on adenovirus type 5, adeno-associated virus type 8, and gp64-pseudotyped HIV lentivirus and, to a lesser extent, after nonviral polyethylenimine (PEI)-DNA delivery. Detection increased relative to the concentration of luciferin; however, a standard concentration of 15 mg/ml was well beyond the saturation point. Compared with intraperitoneal injection, intranasal instillation yields about a 10-fold increase in sensitivity with an approximate 30-fold reduction in luciferin usage when bioimaging in the nasal and pulmonary airways of mice.