Twenty neuropeptide FF (NPFF) analogs having various lengths were synthesized by solid-phase peptide synthesis to gain more information on the role of N-terminal residues for the NPFF receptor affinity. The affinities were evaluated in the rat spinal cord membrane preparations, and the biological activities were measured on morphine analgesia in the mouse tail-flick test. Shortening of the NPFF sequence from the N-terminal produced only a moderate decrease in affinity until NPFF (4-8) was reached. In the same way, NPFF(3-8) significantly decreased morphine analgesia, while NPFF(4-8) had no significant effect at a dose of 22 nmol. The introduction in the N-terminal part of NPFF of a D-enantiomer at positions 2 and 1 or the presence of an N-methyl group on position 3 did not modify affinity and activity. Substitution of proline5 by the D-isomer decreased the affinity of NPFF analogs whatever their length, and [Tyr1,D-Pro5]NPFF(1-8) was 2.5-fold less potent than [Tyr1]NPFF(1-8) in reversing morphine-induced analgesia. In contrast, the presence of a glycine residue in position 5 did not influence the affinity toward NPFF receptors. Data provide evidence that the N-terminal segment of neuropeptide FF is responsible for high-affinity binding.