In order to explore which amino acids or which blocks of amino acids in the 29 amino acid neuropeptide galanin are important for recognition of the endogenous ligand by galanin receptor subtypes present in the jejunum and in the hypothalamus, respectively, we have carried out L-Ala substitutions of individual amino acids or of blocks of amino acids in the rat galanin sequence and examined the binding of the obtained analogs to the rat hypothalamic and jejunal galanin receptor subtypes. This study reveals that the galanin sequence YLLGPH9-14 is essential for recognition of galanin by both the rat hypothalamic and jejunal galanin receptor subtypes. Substitution of the N-terminal amino acids. GWTL1-4, leads to total loss of affinity of galanin for both hypothalamic and jejunal galanin receptors. The alpha-helical C-terminal amino acid (25-29) part of galanin has no greater influence on the affinity of galanin to the hypothalamic galanin receptor subtype. L-Ala substitution of the C-terminal amino acids of galanin KHGLT25-29 shows, however, that this C-terminal motif is essential for the recognition by the jejunal galanin receptor subtype, whereas amino acids in the middle portion of galanin NSAG5-8 are of importance for binding to the hypothalamic but not to the jejunal receptor. [Ala5-8]Galanin thus has a more than 100-fold higher affinity to jejunal receptor than to the hypothalamic receptor, while [Ala25-29]galanin has a more than 100-fold higher affinity for the hypothalamic than for jejunal galanin receptor subtypes. pH dependence of the galanin binding to these receptor subtypes is also different.