In this study, we examined the antinociceptive effect of β-lactotensin, a neurotensin agonist that has been isolated from the chymotrypsin digest of β-lactoglobulin as an ileum-contracting peptide. β-Lactotensin showed naloxone-insensitive antinociceptive activity by the tail-pinch test after i.c.v. (200 nmol/mouse) or s.c. (300 mg/kg) administration in ddY mice. Tolerance was not developed to antinociception induced by β-lactotensin after repeated s.c. administration for 5 days. The antinociceptive activity of β-lactotensin was blocked by treatment with the neurotensin NT 2 receptor antisense ODN, while treatment with the NT 1 receptor antisense ODN had no effect. The antinociceptive activity was also blocked by a dopamine D 1 receptor antagonist, SCH23390 (1 μg/mouse, i.c.v.), while a D 2 receptor antagonist, raclopride (0.5 μg/mouse, i.c.v.), did not block the activity. These results indicate that the antinociceptive activity of β-lactotensin is mediated by NT 2 and D 1 receptors.