The presence of cholinergic nerves in cerebral arteries of several species was investigated by an immunohistochemical method using antibodies against choline acetyltransferase (ChAT). In cats, pigs, rats, and dogs, ChAT immunoreactivities were found to be associated with large bundles and single fibers in the circle of Willis and anterior cerebral, middle cerebral, and basilar arteries. In the rabbit, the ChAT-immunoreactive (ChAT-I) nerves were also observed in the circle of Willis and anterior and middle cerebral arteries, but only few or none were found in the basilar and vertebral arteries. The ChAT-I nerves were found only in the adventitial layer of vessels examined. Superior cervical ganglionectomy did not appreciably affect the distribution of ChAT-I nerves. These results indicate the presence of cholinergic nerves in cerebral arteries. The distribution pattern of ChAT-I nerves was different from that of vasoactive intestinal polypeptide (VIP)-like-immunoreactive nerves and acetylcholinesterase-positive nerves. The possible coexistence of ChAT and VIP-like substance in the same neuron is discussed.