A dose‐dependent increase in tone and reduction in amplitude of contractions was observed after serotonin (5‐HT) was administered to isolated segments of rat ileum, incubated in Locke's solution at 38°C. Melatonin (M) reduced the tone but not the amplitude or frequency of contractions. Addition of M (administered in doses 20 to 100 × higher than 5‐HT) relieved the spasm induced by 5‐HT. Furthermore, pretreatment with M significantly reduced the 5‐HT effect. N‐acetylserotonin (NAS) exhibited delayed but similar effects to M. Neither M nor NAS could prevent or relieve acetylcholine‐induced contractions or influence relief of intestinal contractions by adrenaline. This indicates that 5‐HT and M act via a different mechanism than that of adrenaline and acetycholine system. Serotonin muscle receptor blocker methysergide reduced 5‐HT effect but was not able to abolish it completely. As methysergide could not reduce the muscle tone and did not relieve spasm caused by 5‐HT, it is speculated that M is not acting as antagonist of 5‐HT‐ stimulatory receptors but rather as agonist of 5‐HT‐