The mode of action of serotonin on isolated longitudinal muscle strip with attached Auerbach plexus and on whole ileum preparation has been studied. Atropine, tetrodoxtoxin, morphine and alpha-adrenoceptor stimulants inhibited the contractions of the strips and whole ileum in response to serotonin. In the presence of atropine (2 X 10(-8) M) at 9 times higher concentration of serotonin was needed to produce the same contraction of the ileum as in control experiments. These facts indicate that the contractile effect of serotonin on the longitudinal muscle of ileum might be mediated via a cholinergic pathway. Direct evidence has also been provided that serotonin is capable of releasing acetylcholine from the Auerbach plexus. It is very likely that serotonin stimulates ganglion cells, which in fact leads to firing and, consequently, to an increase of acetylcholine release from the nerve terminals.