Structure-activity relationship (SAR) studies have revealed that the first three residues of galanin (Gly1-Trp2-Thr3) are of critical importance for high-affinity binding to the galanin receptor. Furthermore degradation studies have shown that galanin is easily cleaved to yield inactive fragments in rat hypothalamus (t1/2 = 100 min). To obtain galanin receptor ligands with long-lasting biological activity the amino-terminus of galanin must be protected. We have therefore synthesized analogs of rat galanin(1-16) carrying modifications at the three amino-termini of galanin. All modifications of the peptide backbone flanking Trp2 as in the analogs [N-Me-Trp2]-galanin(1-16), [Tcc2]-galanin-(1-16), (Trp2-psi[CH2NH]-Thr3)-galanin-(1-16) produced a dramatic loss of affinity toward the galanin receptor. [N-Me-Thr3]-galanin(1-16) was the most active of the peptide backbone modified analogs (KD = 997 +/- 1 nM). Modifications of the indole ring in Trp2 ([For-Trp2]-galanin-(1-16), [Tcc2]-galanin-(1-16)) yielded analogs which, at concentrations up to 10 microM, did not displace [125I]galanin binding. N-Methylation of Gly1 by the introduction of sarcosine ([Sar1]-galanin(1-16)) did not significantly affect the ligand-binding properties of galanin(1-16) (KD = 8.7 +/- 0.1 nM).