The pattern of innervation in the aganglionic colon and internal anal sphincter from cogenitally aganglionic rats was studied and compared with that of control littermates. In normoganglionic colon and anal sphincter, electrical stimulation evoked excitatory or inhibitory junction potentials followed by a contraction or relaxation, respectively. These responses were abolished by tetrodotoxin and atropine selectively abolished the excitatory effects, indicating that the colon or anal sphincter is innervated by intrinsic cholinergic excitatory and noncholinergic inhibitory nerves. In congenitally aganglionic rats, electrical stimulation evoked excitatory and inhibitory responses in sphincteric regions, while only excitatory responses were observed in distal segments. Excitatory responses were weak in proximal segments of the aganglionic colon and electrical stimulation failed to evoke neurogenic responses. These results indicate regional differences in the functional innervation of extrinsic nerve fibers in the aganglionic colon from congenitally aganglionic rats and the usefulness of congenitally aganglionic rats as an animal model for Hirschsprung's disease.