The expression of mRNA for the calmodulin-dependent form of brain nitric oxide synthase (NOS) was examined in cholinergic cells of the rat brain using a method combining in situ hybridization histochemistry with immunocytochemistry for choline acetyltransferase (ChAT) in the same brain sections. We constructed a riboprobe specific for brain NOS by subcloning a 493 bp fragment of the coding region which displayed low homology to other forms of NOS. The general distribution of NOS mRNA was in excellent agreement with previous studies using the full-length probe or NADPH diaphorase histochemistry. NOS mRNA was observed in many brain structures and relative levels were quantitated using grain counting procedures in a number of cholinergic and non-cholinergic neuronal groups throughout the brain. In the forebrain, ChAT-immunoreactive cells or cell groups were observed in medial septum (MS), vertical limbs of diagonal band (DBV) and horizontal limbs of diagonal band (DBH), nucleus basalis magnocellularis (NBM), substantia innominata (SI), and striatum (ST). In the brainstem, the cholinergic groups studied included those located in the pedunculopontine tegmental nucleus (PPTN), the laterodorsal tegmental nucleus (LDTN), the nucleus parabigeminalis and several motor nuclei. For NOS mRNA quantitation, silver grains overlying ChAT-stained neuronal profiles in sections on emulsion-dipped slides were counted digitally. In the LDTN and PPTN, virtually all the ChAT-positive cells expressed NOS mRNA at high levels. In MS, DBV and SI, about 30-50% of the ChAT-positive cells expressed NOS mRNA at low-to-moderate levels. Less than 20% of ChAT-positive neurons in the other cholinergic populations studied expressed NOS mRNA; the NBM was one of these low-expressing populations. Many scattered non-cholinergic cells expressing NOS mRNA were found in the striatum and cerebral cortex. In other non-cholinergic regions, high NOS mRNA expression was observed in the islands of Calleja, thalamic and hypothalamic nuclei, several amygdaloid nuclei, regions related to the optic tract, the interpeduncular nucleus, and the supramammillary nucleus. The heterogeneous distribution of NOS mRNA implies complex roles for nitric oxide neurotransmission in brain function, including for the cholinergic phenotype. Additionally, given the postulated involvement of nitric oxide in neurodegeneration, the widely varying levels of expression of NOS within identified central cholinergic neurons may relate to differential vulnerability of this phenotype in disease or aging.