RNA interference (RNAi) is an important part of the cell's defenses against viruses and other foreign genes. Moreover, the biotechnological exploitation of RNAi offers therapeutic potential for a range of diseases for which drugs are currently unavailable. Unfortunately, the small interfering RNAs (siRNAs) that are central to RNAi in the cytoplasm are readily degradable by ubiquitous nucleases, are inefficiently targeted to desired organs and cell types, and are excreted quickly upon systemic injection. As a result, local administration techniques have been favored over the past few years, resulting in great success in the treatment of viral infections and other respiratory disorders. Because there are several advantages of pulmonary delivery over systemic administration, two of the four siRNA drugs currently in phase II clinical trials are delivered intranasally or by inhalation. The air-blood barrier, however, has only limited permeability toward large, hydrophilic biopharmaceuticals such as nucleic acids; in addition, the lung imposes intrinsic hurdles to efficient siRNA delivery. Thus, appropriate formulations and delivery devices are very much needed. Although many different formulations have been optimized for in vitro siRNA delivery to lung cells, only a few have been reported successful in vivo. In this Account, we discuss both obstacles to pulmonary siRNA delivery and the success stories that have been achieved thus far. The optimal pulmonary delivery vehicle should be neither cytotoxic nor immunogenic, should protect the payload from degradation by nucleases during the delivery process, and should mediate the intracellular uptake of siRNA. Further requirements include the improvement of the pharmacokinetics and lung distribution profiles of siRNA, the extension of lung retention times (through reduced recognition by macrophages), and the incorporation of reversible or stimuli-responsive binding of siRNA to allow for efficient release of the siRNAs at the target site. In addition, the ideal carrier would be biodegradable (to address difficulties with repeated administration for the treatment of chronic diseases) and would contain targeting moieties to enhance uptake by specific cell types. None of the currently available polymer- and lipid-based formulations meet every one of these requirements, but we introduce here several promising new approaches, including a biodegradable, nonimmunogenic polyester. We also discuss imaging techniques for following the biodistribution according to the administration route. This tracking is crucial for better understanding the translocation and clearance of nanoformulated siRNA subsequent to pulmonary delivery. In the literature, the success of pulmonary siRNA delivery is evaluated solely by relief from or prophylaxis against a disease; side effects are not studied in detail. It also remains unclear which cell types in the lung eventually take up siRNA. These are critical issues for the translational use of pulmonary siRNA formulations; accordingly, we present a flow cytometry technique that can be utilized to differentiate transfected cell populations in a mouse model that expresses transgenic enhanced green fluorescence protein (EGFP). This technique, in which different cell types are identified on the basis of their surface antigen expression, may eventually help in the development of safer carriers with minimized side effects in nontargeted tissues.