The cellular distribution of adenosine A2A receptor messenger RNA in the central nervous system was investigated using in situ hybridization with ribonucleotide probes. A specific expression was found in the dorsal (i.e. caudate putamen) and ventral (i.e. nucleus accumbens and olfactory tubercle) striatum, the lateral septum and in some cerebellar Purkinje cells. Simultaneous detection of radioactive and non-radioactive probes showed that the majority of adenosine A2A receptor messenger RNA-containing neurons in the dorsal and ventral striatum co-expressed dopamine D2 receptor messenger RNA and preproenkephalin A messenger RNA. However, a minor sub-population of neurons expressing adenosine A2A receptor messenger RNA, but not preproenkephalin A messenger RNA, was found in clusters along the ventral border of the nucleus accumbens. Only a small number of striatal neurons expressing dopamine D1 receptor or substance P messenger RNAs also expressed adenosine A2A receptor messenger RNA. Finally, in the ventral part of nucleus accumbens and in the olfactory tubercle a major sub-population of neurons expressed preproenkephalin A messenger RNA, but not adenosine A2A receptor messenger RNA. Cholinergic interneurons did not express adenosine A2A receptor messenger RNA. Thus, the extensive co-localization of adenosine A2A and dopamine D2 receptors previously described in the dorsal striatum extends into its ventral part. There is also a high degree of co-expression of adenosine A2A receptor messenger RNA and preproenkephalin A messenger RNA in the ventral striatum, but within this region several topologically defined sub-populations of neurons express only one of these transcripts. A majority of the adenosine A2A receptor messenger RNA-containing neurons in the lateral septum did contain preproenkephalin A messenger RNA, whereas only a few co-expressed dopamine D2 receptor messenger RNA. This detailed investigation demonstrates that most of the subcortical areas innervated by dopamine have an abundant, although restricted expression of the adenosine A2A receptor gene and that this receptor is expressed in very few cells outside these areas. These results predict that adenosine A2A receptors are involved not only in motor behaviour, but also in goal-oriented behaviours.