Nitric oxide, the gaseous neuronal messenger, is produced by nitric oxide synthase (NOS) in a Ca2+ dependent manner. Indirect evidence suggests that glutamate, acting through the NMDA subtype of excitatory amino acid receptor, is the principle activator signal for NOS in the brain. To date there is a lack of direct evidence demonstrating NOS and NMDA receptors, or receptor message, in the same neurons. A double labeling technique was developed which uses NOS immunocytochemistry in combination with in situ hybridization for NMDA NR1 receptor mRNA. Quantitative analysis of the silver grain labeling resulting from the hybridization procedure revealed that the majority of NOS positive cells in the cerebral cortex, striatum and midbrain contained a significantly greater amount of NR1 receptor mRNA than non-NOS neurons in the same regions. The amount of NR1 mRNA per cell varies in the cortical and striatal NOS cells, with some cells showing no NR1 expression. These results indicate that NOS containing neurons do not reflect a single phenotype of mRNA expression and further suggest that some NOS cells may be activated by a non-NMDA receptor mechanism.