The nuclei of the auditory brainstem harbor a diversity of neuronal cell types and are interconnected by excitatory as well as inhibitory ascending, descending, and commissural pathways. Classically, neurons have been characterized by size and shape of their cell body and by the geometry of their dendrites. Our study is based on the use of axonal tracers in combination with immunocytochemistry to identify and distinguish neuronal subtypes by their molecular signature in dorsal and ventral cochlear nucleus, lateral superior olive, medial superior olive, medial nucleus of the trapezoid body, and inferior colliculus of the adult rat. The presumed neurotransmitters glutamate, glycine, and GABA were used alongside the calcium-binding proteins parvalbumin, calretinin, and calbindin-D28k as molecular markers. Our data provide distinct extensions to previous characterizations of neuronal subtypes and reveal regularities and differences across auditory brainstem nuclei that are discussed for their functional implications.