The use of traditional neuroanatomic tracing methods with tracers such as horseradish peroxidase requires living systems to take up and transport the label. These tracers have limited application in the study of prenatal systems. The advent of the carbocyanine dye DiI provided a tool by which neuronal pathways may be traced in postmortem fixed tissue. This dye allows careful dissection of prenatal organisms and specific application of the tracer to the neuroanatomic structure under investigation. Although DiI has become increasingly popular, it is limited by the difficulty in applying crystals to peripheral nerves and in the lengthy trace times, particularly in fixed tissue. A chemically modified version of DiI has been introduced that may overcome some of the limitations in using this tracer. The newer dye, fast-DiI, is easy to apply and can trace neural pathways in a shorter period. We describe our experience with the use of fast-DiI in the prenatal rat for the investigation of motoneurons that subserve upper respiratory tract structures. We have determined protocols for fixation, application of dye, processing of tissue, and visualization of traces. The entire protocol can be completed within 1 week, and the use of fast-DiI is easy to learn and apply. The resultant labeling of traced nerves is specific and clearly demonstrates respective motor nuclei and individual motoneurons.