Substance P caused marked analgesic activity in rats after intraventricular administration and in mice after intraperitoneal injection. The hexapeptide pGlu6(SP6-11) was active in mice, but not in rats. Depletion of serotonin with p-chlorophenylalanine abolished the antinociceptive activity in mice, but not in rats, whereas lesion of raphe nuclei blocked the activity of substance P in the latter animals. Although different routes of administration were used, the results seem to indicate different mechanisms of analgesic activity of both peptides in rats and mice, as well as the different role of serotonergic transmission in pain control mechanisms in both species.