An abundance of glycine and glycine receptor immunoreactivities was found in all three parts of the deep cerebellar nuclei. Glycine immunoreactivity was restricted to small neurons throughout most of the deep cerebellar nuclei except for a few large positive neurons in the ventral part of the fastigial nuclei. In addition, glycine immunoreactivity was found in boutons outlining somata of large glycine negative neurons. Complementary to the glycine positive boutons was an intense glycine receptor immunoreactivity on large deep cerebellar nuclei neurons. Comparisons of immunoreactivities for glycine, GABA and aspartate in consecutive one micron sections revealed that many small neurons colocalized glycine and GABA, while some large neurons in the fastigal region colocalized glycine and aspartate. Ultrastructural investigations revealed glycine receptors on postsynaptic sites of dendrites and somata. Most boutons, which were presynaptic to glycine receptor sites, were filled with small flattened vesicles; however, a small percentage of boutons had round clear or dense core vesicles. Frequently, each bouton apposed multiple active zones on the dendrite or soma. One of these active zones was positive for glycine receptor and another was negative. This study supports: (1) glycine as a neurotransmitter in deep cerebellar nuclei, and (2) glycine and GABA colocalization in the same cell and bouton, but releasing to different receptor sites on the target neuron. Furthermore, the coexistence of glycine with GABA in the same deep cerebellar neuron may play an important role in controlling the conset and duration of signal transmission.