The innervation of cerebral blood vessels by nerve fibers containing acetylcholinesterase (AChE) and vasoactive intestinal peptide (VIP) and the vasomotor effects of the two neurotransmitters have been analyzed in the rat following the uni- or bilateral removal of the sphenopalatine ganglion (SPG), which is thought to be the major origin of this innervation. Histochemistry of AChE-positive nerve fibers and the immunoreactivity toward VIP revealed only a 30% reduction in the innervation pattern of the rostral part of the cerebral circulation following the operation. At approximately 4 weeks postoperatively, the original nerve network was restored. Quantitative measurements of cholineacetyltransferase activity and VIP revealed similar reductions in the levels of collected large cerebral arteries at the base of the brain and in small pial vessels overlying the cerebral cortex at the various postoperative times following uni- or bilateral removal of the SPG. The two techniques thus complemented each other. Vasomotor reactivity to acetylcholine (ACh) and VIP was examined in proximal segments of the middle cerebral artery at the various postoperative times. Generally, the removal of the SPG had no effect on the responses to ACh or VIP. The evidence indicates that only approximately one-third of the cholinergic/VIP innervation of the rostral part of the cerebral circulation originates in the SPG.