The expression of hippocampal cholinergic neurostimulating peptide (HCNP)-precursor mRNA in rat brain was examined by Northern blot and in situ hybridization analyses. Northern blot analysis using rat HCNP-precursor cDNA revealed a 1.1-kilobase (kb) transcript. A message of identical size was also detected with the antisense precursor riboprobe. In situ hybridization disclosed that HCNP-precursor mRNA was expressed in many areas of the brain, including the basal forebrain cholinergic system, the olfactory system, and the cerebellum. Very high levels were seen in the pyramidal cells of the CA3 region and in the hilus of the dentate gyrus of the hippocampal formation. High levels were also found in the septal area, piriform cortex, entorhinal cortex, thalamic nuclei, subthalamic nuclei, medial habenular nuclei, substantia nigra, Purkinje cells of the cerebellum, and choroid plexus. By contrast, glial cells were not labeled by the antisense HCNP-precursor riboprobe. The expression of HCNP-precursor mRNA by a variety of neurons suggests that HCNP and its precursor protein play significant roles in the stimulation of cholinergic activity, as well as in other not yet defined functions.