The acetylcholinesterase (AChE) content of rat vagal efferent neurons was studied. Retrograde transport of horseradish peroxidase (HRP) by cut vagal axons provided a means for localizing efferent cell bodies; tissue sections were then processed for the simultaneous visualization of HRP and AChE. A dorsal vagal efferent column contained the dorsal motor nucleus of the vagus, as a primary component, and extended caudally into the upper cervical spinal cord. A ventral column contained neurons in the nucleus ambiguus and the surrounding reticular formation. Although most of the vagal efferent neurons stained with moderate to heave intensity for AChE there were some HRP-labeled cells that contained little AChE and a small percentage in which AChE was absent. In spite of the fact that AChE has been demonstrated in certain non-cholinergic neurons, it has also been found in all cholinergic neurons. Therefore, the presence of AChE has been regarded as a necessary (but not sufficient) component for identifying cholinergic neurons. The absence of AChE in a small percentage of the vagal efferent neurons indicates that some preganglionic parasympathetic fibers in the vagus nerve are not cholinergic.