Using in situ hybridization with multiple probes to the rat preproenkephalin gene, we have identified a novel population of cells in the reticular thalamic nucleus and basal forebrain which express RNA derived from the preproenkephalin gene. These cells contain nuclear RNA from downstream of an alternate transcription start site in intron A of the preproenkephalin gene (Kilpatrick et al., Mol. Cell Biol., 10 (1990) 3717-3726), while in the same cells preproenkephalin exon 2 RNA is undetectable. The results suggest that in this population of cells, preproenkephalin gene transcription initiates from the intron A initiation site, and is regulated by an additional mechanism which results in the accumulation of nuclear preproenkephalin intron A-derived heteronuclear RNA. The anatomical distribution of these cells indicates that they may be involved in the control of cerebral cortical function.