We are investigating the nature of the chemical interactions between the neuropeptide Y (NPY) and its cell surface receptor (Y1). A previous study involving site-directed mutagenesis and computer-aided modelling (Walker et al., 1994) suggested that the C-terminal Tyr36 of NPY, known to be a key residue for receptor binding, might dock at a pocket formed by hydrophobic amino acids of transmembrane domains (TM) 1, 2, 6 and 7 of the Y1 receptor. To investigate which residues were required for ligand binding, we mutated the sequences encoding F41, L43, F96, Y100, F286 and H298 of the human Y1 receptor. The mutant cDNAs were transiently expressed in Hela cells and the ability of the encoded proteins to bind NPY was evaluated. Replacing F41, L43 or F96 with alanines had no effect on NPY binding. On the contrary, Y100, F286 and H298 appeared to be residues critical for ligand binding. In particular, the removal of the hydroxyl group of Y100 (Tyr100-->Phe100 mutation) yielded a protein devoid of affinity for the ligand. The level of expression and the presence on the cell surface of mutants lacking NPY binding activity was assessed by immunological techniques. In addition, we tested the ability of synthetic analogues of neuropeptide Y with substitutions at position 36 to bind to the Y1 receptor. To get spatial insight into the relative positions of the above mentioned residues we constructed a molecular model of the interaction between NPY:Y36 and the elements of the hydrophobic pocket surrounding this residue.