Acetylcholine (ACh) regulates arousal and the present study was designed to provide insight into the neurochemical mechanisms modulating ACh release in the pontine reticular formation. Nitric oxide-releasing beads microinjected into the pontine reticular formation of C57BL/6J (B6) mouse caused a significant (p<0.0001) increase in ACh release. Microdialysis delivery of the nitric oxide donor NOC-12 to the mouse pontine reticular formation also caused a concentration dependent increase in ACh release (p<0.001). These are the first neurochemical data showing that ACh release in pontine reticular formation of B6 mouse is modulated by nitric oxide. The signal transduction cascade through which nitric oxide modulates ACh release in pontine reticular formation has not previously been characterized. Therefore, an additional series of studies quantified the effects of a soluble guanylate cyclase (sGC) inhibitor, ODQ, on ACh release in cat medial pontine reticular formation. During naturally occurring states of sleep and wakefulness, but not during states of anesthesia, ODQ caused a significant (p<0.001) decrease in ACh release. These results show for the first time that nitric oxide modulates ACh in cat medial pontine reticular formation via a nitric oxide-sensitive, soluble guanylate cyclase signal transduction cascade. Isoflurane and halothane anesthesia previously have been shown to decrease ACh release in the medial pontine reticular formation. The finding that ODQ did not alter ACh release during isoflurane or halothane anesthesia demonstrates that these anesthetics disrupt the nitric oxide-sensitive, sGC-guanosine 3',5'-cyclic monophosphate (cGMP) pathway. Considered together, the results from both mouse and cat indicate that nitric oxide modulates ACh release in arousal-promoting regions of the pontine reticular formation via a nitric oxide-sensitive, sGC-cGMP pathway.