High-affinity transporters mediate the removal of released neurotransmitters from synapses, thereby terminating their synaptic action. A novel glycine transporter has recently been cloned from a rat brain complementary DNA library. In this study we examined, by means of in situ hybridization with 35S-labelled oligodeoxynucleotide probes, the distribution of messenger RNAs encoding glycine transporter 2 in the rat CNS. Moreover, adjacent series of sections were labelled with [3H]strychnine to reveal the regional distribution of strychnine-sensitive glycine receptors. A very discrete pattern of distribution of the transcripts was found exclusively at the level of the brainstem/cerebellum and spinal cord. In the cerebellum, Golgi cells in the granule cell layer as well as a subpopulation of neurons in the interposed nuclei were consistently labelled. In the brainstem, where the bulk of the labelling was concentrated, several nuclei showed a high level of transcript expression, including the superior olivary complex, nucleus of the trapezoid body and the ventral nucleus of the lateral lemniscus. In the spinal cord, many neurons throughout all layers were labelled, including putative Renshaw cells and a few large neurons at the border of layers 7 and 9. No labelled cells were detected at the levels of the fore- and midbrain. The distribution of glycine transporter 2 messenger RNA-containing cell bodies was very different to that of other glycine transporter messenger RNAs (glycine transporter 1a and glycine transporter 1b), but similar to that of known glycine-immunoreactive neurons and correlated very well with that of strychnine-sensitive glycine receptors in most CNS regions except cerebellum. Our results show that glycine transporter 2 (but not glycine transporter 1) in the brainstem, spinal cord and cerebellum is probably involved in the reuptake of glycine from synapses containing classical strychnine-sensitive glycine receptors. Our findings also suggest that glycine acts as a neurotransmitter in cerebellar Golgi neurons. Whether the synaptic concentration of glycine, as co-agonist at NMDA receptors, is regulated (if at all) by transaminase activity or by a glycine transporter (GLYT1a?) distinct from that described here is not yet known.