On the smooth muscle of the gastrointestinal system, substance P (SP) has both a stimulatory and an inhibitory effect. The effect depends on dose and route of application (in vivo or in vitro). In vivo (stomach and ileum of dog) at low dose, SP inhibits the amplitude and frequency of peristaltic contractions without influencing tone. In higher dose, SP stimulates the tone and inhibits peristaltic contractions. In vitro (circular preparations of fundus and corpus of guinea pig stomach and longitudinal preparations of guinea pig ileum), only the stimulatory effect of SP is found. At high SP concentrations, stimulation of tone is accompanied by a postexcitatory inhibition of peristaltic contractions. The results demonstrate that there are two types of inhibitory activity of SP on contractions of smooth muscles. One of these can be seen only during in vivo experiments at low concentrations. This primary inhibitory effect could be the result of the action of SP on the neuronal elements. The other kind of inhibitory effect on contraction is a secondary effect, and is only induced at high concentrations of SP and only in connection with a stimulatory effect on tone. The secondary inhibitory effect of SP may be connected with the direct effect of SP on smooth muscle cells.