The neural pathway involved in activation of the tensor tympani (TT) muscle was studied in the rat using retrograde HRP and transneuronal viral tracing techniques. The pool of TT motoneurons labeled with HRP was located ipsilaterally under the anterior third of the trigeminal motor nucleus and extended rostrally towards the lateral lemniscus. The origin of the inputs to these motoneurons was then determined using transneuronal viral transport: presumably transneuronally infected neurons appeared bilaterally in the vicinity of the superior olivary complex, mainly in between the two nuclei of the trapezoid body. The present data are consistent with previous conclusions based on lesion experiments that the TT reflex loop is made up of a chain of 4 neurons.