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Neurotensin and several sequence analogues have been synthesized using solid-phase technology. The purity of the following derivatives: neurotensin, neurotensin-(10–13), neurotensin-(9–13). neurotensin-(8–13), neurotensin-(6–13), neurotensin-(4–13), [Cit8]neurotensin-(8–13), [Lys8]neurotensin-(8–13), [Cit9]neurotensin-(8–13),[Lys9]neurotensin-(8–13), [Phe11]neurotensin-(8–13), [Ala12]neurotensin-(8–13) and [Ala13]- neurotensin-(8–13) was verified by amino acid analyses after acid and enzymatic hydrolyses. reverse-phase high- performance liquid chromatography in two systems and Edman degradation. The above analogues, those obtained after N-acetylation of neurotensin-(6–13), neurotensin-(8–13), [Cit8]neurotensin-(8–13), [Cit9]- neurotensin-(8–13), [Lys8]neurotensin-(8–13), [Lys9]neurotensin-(8–13) and [Phe11]neurotensin-(8–13), as well as native xenopsin, were all tested for binding competition with [3H]neurotensin on the specific fixation sites of rat brain synaptosomal membranes and on those of HT 29 cells. In addition to these radioreceptor assays on neural and extraneural targets, a pharmacological test (contraction of guinea pig ileum in the presence of neostigmine) was used to compare the behavior of the synthetic analogues. The use of these three biological systems enabled us to obtain consistent results. A good parallel was observed between the degree of fixation and pharmacological effects for entire neurotensin and for C-terminal region analogues up to the size of neurotensin- (8–13). The two peptides neurotensin-(6- 13) and neurotensin-(4–13) had an abnormally high affinity for rat brain synaptic membrane binding sites compared to a relatively low contracting activity. The C-terminal peptide -Arg-Arg-Pro-Tyr-Ile-Leu fulfills all the structural requirements for mimicking the entire sequence, provided its α-amino end is protected by acetylation. The guanidinium structure of residues 8 and 9 are not of vital importance, since they could be efficiently replaced by amino groups of lysyl side chains. Xenopsin, which can be considered as a natural analogue of neurotensin-(8–13), acts similarly to acetyl-neurotensin-(8–13). Removal of the phenolic function of residue 11 induces a decrease in neurotensin effects. The C-terminal isoleucyl and leucyl residues could not be replaced by alanine without complete loss of the three activities tested.