Cyclic nucleotides are major intracellular mediators in the signal transduction events in synaptic neurotransmission of the CNS. Intracellular Ca2+ is known to regulate adenylyl cyclase (AC) in a calmodulin (CaM)-dependent manner, and guanylyl cyclase (GC), in an indirect manner through CaM-sensitive nitric oxide synthase. To ascertain the physiological significance of cyclic nucleotide second messenger systems, we have localized the mRNAs encoding AC, GC, and CaM in the rat brain by in situ hybridization using 35S-labeled RNA probes. The AC mRNA is widely distributed throughout the brain; strong hybridization signal was observed in the granular layers of the cerebellum, in the pyramidal and granule cells of the hippocampus, and in the olfactory system. These AC mRNA localizations are compatible with the distribution of Ca2+/CaM-sensitive AC activities. In contrast to AC mRNA distribution, GC mRNA has a more limited distribution. Significant signals were observed in the striatum, in the pyramidal and granule cells of the hippocampus, in the olfactory system, in the inferior and superior colliculus, in the Purkinje cells of the cerebellum, in the locus coeruleus, and in many pyramidal cells in the layers II-III and V of the cerebral cortex, and mainly, in the occipital cortex. In some discrete brain regions, a close correlation was found between enzyme activity and mRNA hybridization signal of GC. The distinct distribution of AC and GC mRNAs suggests that different cyclic nucleotide second messenger systems have specialized functions. On the other hand, CaM mRNA was colocalized with the AC and GC mRNA, but its distribution was more abundant and specific for neuronal cells, since there was little hybridization signal with CaM probe in neuronal fiber regions such as the corpus callosum and the anterior commissure. The high expression of CaM mRNA in neuronal cells is in agreement with its biochemical role in the regulation of various enzymes. Results of the present study should help in analyzing the role of cyclic nucleotides and CaM in physiological and pathological situations in the CNS.