We describe a method for applying the carbocyanine dye DiI to the rat heart that takes advantage of the dye's lipophilic properties and its ability to diffuse easily into tissues, and results in specific retrograde labelling of cardiac vagal preganglionic neurones in the medulla oblongata. Most of the labelled neurones were found bilaterally in the nucleus ambiguus (81%), with a few sparsely distributed in the dorsal motor vagal nucleus (6.5%), and in an intermediate area located between these two nuclei (12.5%). We contend that the method of applying DiI crystals to the surface of the heart is a more efficient, accurate and reproducible method of retrograde labelling than the injection of tracers into this very delicate tissue.