The binding and biological activities of neurotensin and two analogues, [TRP11]-neurotensin and xenopsin, in which a tryptophan replaces the neurotensin residue Tyr11, were compared in rat and guinea-pig. The binding activity of three peptides was measured as their ability to inhibit the binding of [3H]neurotensin to rat and guinea-pig brain synaptic membranes. Their biological activities were measured as their effects on the contractility of rat and guinea-pig ileal smooth muscle preparations. In binding as well as biological assays, it was found that [Trp11]-neurotensin and xenopsin were as potent as neurotensin in the rat. In contrast, the two analogues were about 10 times less potent than neurotensin in the guinea-pig. These findings reveal differences between rat and guinea-pig neurotensin receptors should be considered when comparing the activity of neurotensin analogues in assays using tissue preparations from various animal species.