The amino acids GABA and glutamate (Glu) are thought to be the principal substances in the central nervous system responsible for neuronal inhibition and excitation. Their distributions among the different neurons in a defined pathway may thus be indicative of the contributions of the cells to pathway function. Examples of such neurons are those of the cerebellar nuclei which, while regulating output from the Purkinje cells of the cerebellar cortex, are also found to project back to the cerebellar cortex. Immunohistochemical experiments were done to identify GABA and glutamate (Glu) containing cells in the adult rat cerebellar nuclei. Consecutive semithin and serial vibratome sections were incubated with antisera raised in rabbit against GABA and Glu. In semithin sections, only small neurons were intensely GABA immunoreactive (GABA-IR) (31.7%), and the majority (80.5%) were Glu immunoreactive (Glu-IR) of different sizes. Consistent with Glu being a metabolic precursor for GABA, 75.4% of the GABA-IR population colocalized Glu. In vibratome sections GABA-IR neurons showed some local differences in number, whereas the Glu-IR were uniformly distributed in the three nuclei studied. Measured mean diameters for these neurons showed a distinct size difference for the GABA- and Glu-IR with little overlap. Cerebellar nuclei neurons projecting to the cortex (nucleocortical neurons, NCN) were identified by locally preinjecting the retrograde transported WGA-apoHRP-colloidal gold complex in the cerebellar cortex. Vibratome sections of these cerebellar were silver intensified for the retrograde tracer and double labeled for GABA and Glu. Of the total number of identified NCN, 8.7% were GABA-IR (10 animals) and 47.7% Glu-IR (5 animals). Many retrograde labeled NCN in the core of the thick sections were immunonegative for both amino acids due to poor antibody penetration, thus underestimating the proportions of cells containing GABA and Glu. The size distributions for the GABA-IR and Glu-IR NCN were similar to those measured in non-retrograde labeled nuclei in thick sections. The conclusions reached are that GABA-IR neurons of the cerebellar nuclei, including the NCN, use GABA as the presumed inhibitory neurotransmitter and that Glu-IR neurons may use Glu or another excitatory neurotransmitter.